Leishmania antigens for use in the therapy and diagnosis of leishmaniasis

ABSTRACT

Compositions and methods for preventing, treating and detecting leishmaniasis and stimulating immune responses in patients are disclosed. The compounds provided include polypeptides that contain at least an immunogenic portion of one or more Leishmania antigens, or a variant thereof. Vaccines and pharmaceutical compositions comprising such polypeptides, or polynucleotides encoding such polypeptides, are also provided and may be used, for example, for the prevention and therapy of leishmaniasis, as well as for the detection of Leishmania infection.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 09/639,206 filed Aug. 14, 2000, which is a continuation-in-partof U.S. patent application Ser. No. 09/565,501 filed May 5, 2000, whichis a continuation-in-part of U.S. patent application Ser. No. 09/551,974filed Apr. 14, 2000, which is a continuation-in-part of U.S. patentapplication Ser. No. 09/183,861, filed Oct. 30, 1998, now U.S. Pat. No.6,365,165, which is a continuation-in-part of U.S. patent applicationSer. No. 09/022,765, filed Feb. 12, 1998, now U.S. Pat. No. 6,375,955,which is a continuation-in-part of U.S. patent application Ser. No.08/920,609, filed Aug. 27, 1997, which is a continuation-in-part of U.S.patent application Ser. No. 08/798,841, filed Feb. 12, 1997, which is acontinuation-in-part of U.S. patent application Ser. No. 08/533,869,filed Sep. 22, 1995, now U.S. Pat. No. 5,834,592.

TECHNICAL FIELD

The present invention relates generally to compositions and methods forpreventing, treating and detecting leishmaniasis, and for stimulatingimmune responses in patients. The invention is more particularly relatedto polypeptides comprising an immunogenic portion of a Leishmaniaantigen or a variant thereof, and to vaccines and pharmaceuticalcompositions comprising one or more such polypeptides. The vaccines andpharmaceutical compositions may be used, for example, for the preventionand therapy of leishmaniasis, as well as for the detection of Leishmaniainfection.

BACKGROUND OF THE INVENTION

Leishmania organisms are intracellular protozoan parasites ofmacrophages that cause a wide range of clinical diseases in humans anddomestic animals, primarily dogs. In some infections, the parasite maylie dormant for many years. In other cases, the host may develop one ofa variety of forms of leishmaniasis. For example, the disease may beasymptomatic or may be manifested as subclinical visceral leishmaniasis,which is characterized by mild symptoms of malaise, diarrhea andintermittent hepatomegaly. Patients with subclinical or asymptomaticdisease usually have low antibody titers, making the disease difficultto detect with standard techniques. Alternatively, leishmaniasis may bemanifested as a cutaneous disease, which is a severe medical problem butis generally self-limiting, or as a highly destructive mucosal disease,which is not self-limiting. Finally, and most seriously, the disease maybe manifested as an acute visceral infection involving the spleen, liverand lymph nodes, which, untreated, is generally a fatal disease.Symptoms of acute visceral leishmaniasis include hepatosplenomegaly,fever, leukopenia, anemia and hypergammaglobulinemia.

Leishmaniasis is a serious problem in much of the world, includingBrazil, China, East Africa, India and areas of the Middle East. Thedisease is also endemic in the Mediterranean region, including southernFrance, Italy, Greece, Spain, Portugal and North Africa. The number ofcases of leishmaniasis has increased dramatically in the last 20 years,and millions of cases of this disease now exist worldwide. About 2million new cases are diagnosed each year, 25% of which are visceralleishmaniasis. There are, however, no vaccines or effective treatmentscurrently available.

Accurate diagnosis of leishmaniasis is frequently difficult to achieve.There are 20 species of Leishmania that infect humans, including L.donovani, L. chagasi, L. infantum, L. major, L. amazonensis, L.braziliensis, L. panamensis, L. mexicana, L. tropica, and L. guyanensis,and there are no distinctive signs or symptoms that unambiguouslyindicate the presence of Leishmania infection. Parasite detectionmethods have been used, but such methods are neither sensitive norclinically practical. Current skin tests typically use whole or lysedparasites. Such tests are generally insensitive, irreproducible andprone to cross-reaction with a variety of other diseases. In addition,the preparations employed in such tests are often unstable. Thus, thereis a need for improved methods for the detection of Leishmaniainfection.

Current experimental vaccines consisting of whole organisms have notproven effective in humans. Accordingly, there remains a need in the artfor vaccines to prevent leishmaniasis in humans and dogs, and forimproved therapeutic compositions for the treatment of leishmaniasis.

SUMMARY OF THE INVENTION

Briefly stated, the present invention provides compositions and methodsfor preventing, treating and detecting leishmaniasis, as well as forstimulating immune responses in patients. In one aspect, polypeptidesare provided which comprise at least an immunogenic portion of aLeishmania antigen, or a variant of such an antigen that differs only inconservative substitutions and/or modifications. In specific embodimentsof the invention, the Leishmania antigen comprises an amino acidsequence selected from the group consisting of SEQ ID NO: 2, 4, 20, 22,24, 26, 36-38, 41, 50-53, 82, 104, 106, 108, 110, 112 and 118-122,. DNAsequences encoding the above polypeptides, recombinant expressionvectors comprising these DNA sequences and host cells transformed ortransfected with such expression vectors are also provided.

In further aspects, the present invention provides fusion proteinscomprising Leishmania antigens, together with polynucleotides encodingsuch fusion proteins. In certain specific embodiments, such fusionproteins comprise an amino acid sequence selected from the groupconsisting of SEQ ID NOs:95, 96, and 97 and encoded by a polynucleotidesequence selected from the group consisting of SEQ ID NOs:94, 98, 99,100, and 101.

In related aspects, the present invention provides pharmaceuticalcompositions which comprise one or more of the polypeptides and/orfusion proteins described herein, or a polynucleotide encoding suchpolypeptides and fusion proteins, and a physiologically acceptablecarrier. Vaccines which comprise one or more such polypeptides, fusionproteins or polynucleotides, together with an immunostimulant are alsoprovided. In specific embodiments of these aspects, the Leishmaniaantigen has an amino acid sequence selected from the group consisting ofSEQ ID NO: 2, 4, 20, 22, 24, 26, 36-38, 41, 50-53, 82, 104, 106, 108,110, 112 and 118-122.

In still further related embodiments, the pharmaceutical compositionsand vaccines comprise at least two different polypeptides, eachpolypeptide comprising an immunogenic portion of a Leishmania antigenhaving an amino acid sequence selected from the group consisting ofsequences recited in SEQ ID NO: 2, 4, 6, 8, 10, 20, 22, 24, 26, 36-38,41, 50-53, 82, 104, 106, 108, 110, 112 and 118-122, and variants thereofthat differ only in conservative substitutions and/or modifications. Inother embodiments, the inventive pharmaceutical compositions compriseone or more of the inventive polypeptides in combination with a knownLeishmania antigen.

In yet other related embodiments, the pharmaceutical compositions andvaccines comprise soluble Leishmania antigens.

In another aspect, the present invention provides methods for inducingprotective immunity against leishmaniasis in a patient, comprisingadministering to a patient a pharmaceutical composition or vaccine asdescribed above.

In further aspects, methods and diagnostic kits are provided fordetecting Leishmania infection in a patient. The methods comprise: (a)contacting dermal cells of a patient with a pharmaceutical compositionas described above; and (b) detecting an immune response on thepatient's skin, therefrom detecting Leishmania infection in the patient.The diagnostic kits comprise: (a) a pharmaceutical composition asdescribed above; and (b) an apparatus sufficient to contact thepharmaceutical composition with the dermal cells of a patient.

In further aspects, the present invention provides methods forstimulating a cellular and/or humoral immune response in a patient,comprising administering to a patient a pharmaceutical composition orvaccine as described above.

In a related aspect, methods are provided for treating a patientafflicted with a disease responsive to IL-12 stimulation, comprisingadministering to a patient a pharmaceutical composition or vaccine asdescribed above.

These and other aspects of the present invention will become apparentupon reference to the following detailed description and attacheddrawings. All references disclosed herein are hereby incorporated byreference in their entirety as if each was incorporated individually.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the stimulation of proliferation of T-cells obtained fromL. donovani-immunized BALB/c mice (represented by stimulation index) byL. donovani-infected macrophages after incubation for 24, 48 and 72hours.

FIG. 2 illustrates representative HPLC profiles of peptides isolatedfrom MHC class II molecules of P388D1 macrophages. Panel A showspeptides isolated from uninfected macrophages and panel B shows peptidesisolated from L. donovani infected macrophages. The arrows in panel Bindicate peptide peaks present only in the infected macrophagepreparation.

FIG. 3 illustrates the expression and purification of the Leishmaniaantigen Ldp23 as a recombinant fusion protein. Panel A shows a Coomassieblue-stained SDS-PAGE gel of lysed E. coli without (lane 1) and with(lane 2) IPTG induction of Ldp23 expression. Arrow indicates therecombinant fusion protein. Panel B shows the fusion protein followingexcision from a preparative SDS-PAGE gel, electroelution, dialysisagainst PBS and analytical SDS-PAGE.

FIG. 4 presents a Northern blot analysis of total RNA prepared from L.donovani, L. major, L. amazonensis and L. pifanoi with a ³²P labeledLdp23 gene. 1, 2 and 3 refer to RNA obtained from promastigotes at thelogarithmic growth phase, promastigotes at the stationary growth phaseand amastigote forms, respectively.

FIG. 5 shows a Western blot analysis of L. donovani promastigoteantigens incubated with pre-immune rabbit serum (lane A) or withanti-Ldp23 rabbit antiserum (lane B).

FIG. 6 illustrates the surface expression of Ldp23 on live L. donovanipromastigotes. The dotted line shows the indirect immunofluorescenceperformed using pre-immune mouse serum and the solid line shows theresult obtained with mouse anti-GST-Ldp23 antiserum. Fluorescenceintensity was analyzed by FACScan.

FIG. 7 shows the stimulation of Leishmania-specific T-cell proliferationby Ldp23. The results are presented as relative cell number as afunction of fluorescence intensity. T-cells (10⁵/well) were purifiedfrom lymph nodes of BALB/c mice immunized in the foot pad with L.donovani promastigotes in CFA and were cultured with variousconcentrations of the purified recombinant Ldp23 in the presence of2×10⁵ Mitomycin C-treated normal BALB/c spleen mononuclear cells.Proliferation of T-cells was measured at 27 hours of culture. Values areexpressed as cpm and represent the mean of [³H]TdR incorporation oftriplicate cultures.

FIG. 8 illustrates Ldp23-induced cytokine production by lymph node cellsof BALB/c mice. Cultures were incubated with varying amounts of Ldp23 orLeishmania lysate, presented as μg/mL, and were assayed by ELISA for theproduction of interferon-γ (panel A) or interleukin-4 (panel B), both ofwhich are shown as ng/mL.

FIG. 9 shows the PCR amplification of cytokine mRNAs isolated frommucosal leishmaniasis (Panel A) and cutaneous leishmaniasis (panel B)patient PBMC before and after stimulation with representativepolypeptides of the present invention. Lanes O and - indicate the levelof PCR products at the initiation of culture and after 72 hours ofculture, respectively, in the absence of added polypeptide; lanes Lb,83a and 83b indicate the level of PCR products following culturing ofPBMC with L. braziliensis lysate, and the Leishmania antigens Lbhsp83aand Lbhsp83b, respectively.

FIG. 10 presents a comparison of the levels of interferon-γ (panel A)and TNF-α (panel B) in the supernatants of 72 hour PBMC cultures fromLeishmania-infected and control individuals in response to stimulationwith parasite lysate or the indicated polypeptides.

FIG. 11 illustrates the levels of IL-10 p40 (in pg/mL) in thesupernatant of PBMC cultures from L. braziliensis-infected individualsand uninfected controls 72 hours following stimulation with parasitepromastigote lysate (Lb), Lbhsp83a or Lbhsp83b.

FIG. 12 presents the reactivities of sera from L. braziliensisinfected-patients with representative polypeptides of the presentinvention in a standard ELISA. Values are expressed as absorbance at 405nm.

FIGS. 13A and 13B illustrate the level of secreted IL-4 and IFN-γ (inpg/mL) stimulated in mouse lymph node cultures by the addition ofrepresentative polypeptides of the present invention.

FIG. 14 shows the level of IFN-γ (in pg/mL) secreted byLeishmania-infected and uninfected human PBMC stimulated by theLeishmania antigen M15, as compared to the levels stimulated by L. majorlysate and L-Rack, an antigen that does not appear to be recognized byLeishmania-infected humans.

FIG. 15 shows the level of IFN-γ (in pg/mL) secreted by infected anduninfected human PBMC stimulated by soluble Leishmania antigens (Santigens), as compared to the levels stimulated by L. major lysate andL-Rack.

FIG. 16 illustrates the proliferation of murine lymph node culturesstimulated by the addition of representative polypeptides of the presentinvention. Values are expressed as cpm.

FIG. 17 shows the proliferation of human PBMC, prepared fromLeishmania-immune and uninfected individuals, stimulated by M15 ascompared to the proliferation stimulated by L. major lysate and L-Rack.Values are expressed as cpm.

FIG. 18 illustrates the proliferation of human PBMC, prepared fromLeishmania-infected and uninfected individuals, stimulated by solubleLeishmania antigens as compared to the proliferation stimulated byculture medium, L. major lysate and L-Rack. Values are expressed as cpm.

FIG. 19 presents a comparison of a Lbhsp83 sequence (SEQ ID NO:6) withhomologous sequences from L. amazonensis (Lahsp83) (SEQ ID NO:16), T.cruzi (Tchsp83) (SEQ ID NO:17) and humans (Huhsp89) (SEQ ID NO:18).

FIG. 20 illustrates the reactivity of rabbit sera raised against solubleLeishmania antigens with Leishmania promastigote lysate (lane 1) andsoluble Leishmania antigens (lane 2).

FIG. 21 shows the cDNA and predicted amino acid sequence for theLeishmania antigen Lmsp1a.

FIG. 22 shows a Southern blot of genomic DNA from L. major digested witha panel of restriction enzymes (lanes 1 to 7) and six other Leishmaniaspecies digested with PstI (lanes 8 to 13) probed with the full-lengthcDNA insert of Lmspla.

FIG. 23 shows a Southern blot of genomic DNA from L. major digested witha panel of restriction enzymes, six other Leishmania species digestedwith PstI and the infectious pathogens T. cruzi and T. brucei, probedwith the full-length cDNA insert of the Leishmania antigen MAPS-1A.

FIG. 24 illustrates the proliferation of PBMC isolated fromuninfected-individuals, patients with active mucosal leishmaniasis andpatients post kala-azar infection, stimulated by MAPS-1A.

FIG. 25 illustrates the proliferation of murine lymph node culturesstimulated by MAPS-1A.

FIG. 26 illustrates the reactivity of MAPS-1A with sera from humanleishmaniasis patients.

FIG. 27 illustrates the reactivity of MAPS-1A with sera from miceimmunized against and/or infected with leishmaniasis.

FIG. 28 illustrates the effectiveness of immunization with eithersoluble Leishmania antigens or a mixture of Ldp23, LbeiF4A and M15 plusadjuvant in conferring protection against infection (as measured byfootpad swelling) in a murine leishmaniasis model system, as compared tothe administration of adjuvant alone.

FIG. 29 illustrates the effectiveness of immunization with MAPS-1A plusadjuvant in conferring protection against infection (as measured byfootpad swelling) in a murine leishmaniasis model system, as compared tothe administration of adjuvant alone.

FIGS. 30A and B illustrate the proliferation of murine lymph nodecultures stimulated with either LcgSP8, LcgSP10 or LcgSP3.

FIG. 31 illustrates the effectiveness of immunization with solubleLeishmania antigens, MAPS-1A and M15 plus adjuvant, IL-12, in conferringprotection against infection (as measured by footpad swelling) in amurine leishmaniasis model system, as compared to the administration ofadjuvant IL-12 alone.

FIG. 32 illustrates the effectiveness of immunization with M15 DNA andMAPS-1A DNA in conferring protection against infection (as measured byfootpad swelling) in a murine leishmaniasis model system, as compared tocontrol DNA and saline.

FIG. 33 illustrates the effectiveness of immunization with Leishmaniafusion proteins plus IL-12 as adjuvant, in conferring protection againstinfection in a murine leishmaniasis model system.

FIG. 34 illustrates the effectiveness of immunization with Leishmaniafusion proteins plus the adjuvant MPL-SE, in conferring protectionagainst infection in a murine leishmaniasis model system.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention is generally directed tocompositions and methods for preventing, treating and detectingleishmaniasis, as well as for stimulating immune responses in patients.The compositions of the subject invention include polypeptides thatcomprise at least an immunogenic portion of a Leishmania antigen, or avariant of such an antigen. In one preferred embodiment, compositions ofthe present invention include multiple polypeptides selected so as toprovide enhanced protection against a variety of Leishmania species.

Polypeptides within the scope of the present invention include, but arenot limited to, polypeptides comprising immunogenic portions ofLeishmania antigens comprising the sequences recited in SEQ ID NO:2(referred to herein as M15), SEQ ID NO:4 (referred to herein as Ldp23),SEQ ID NO:6 (referred to herein as Lbhsp83), SEQ ID NO:8 (referred toherein as Lt-210), SEQ ID NO:10 (referred to herein as LbeIF4A), SEQ IDNO: 20 (referred to herein as Lmspla), SEQ ID NO: 22 (referred to hereinas Lmsp9a), SEQ ID NOs: 24 and 26 (referred to herein as MAPS-1A), andSEQ ID NO: 36-38, 41, 50-53, 82, 104, 106, 108, 110, 112 and 118-122. Asused herein, the term “polypeptide” encompasses amino acid chains of anylength, including full length proteins (i.e., antigens), wherein theamino acid residues are linked by covalent bonds. Thus, a polypeptidecomprising an immunogenic portion of one of the above antigens mayconsist entirely of the immunogenic portion, or may contain additionalsequences. The additional sequences may be derived from the nativeLeishmania antigen or may be heterologous, and such sequences may (butneed not) be immunogenic. An antigen “having” a particular sequence isan antigen that contains, within its full length sequence, the recitedsequence. The native antigen may, or may not, contain additional aminoacid sequence.

An immunogenic portion of a Leishmania antigen is a portion that iscapable of eliciting an immune response (i.e., cellular and/or humoral)in a presently or previously Leishmania-infected patient (such as ahuman or a dog) and/or in cultures of lymph node cells or peripheralblood mononuclear cells (PBMC) isolated from presently or previouslyLeishmania-infected individuals. The cells in which a response iselicited may comprise a mixture of cell types or may contain isolatedcomponent cells (including, but not limited to, T-cells, NK cells,macrophages, monocytes and/or B cells). In particular, immunogenicportions are capable of inducing T-cell proliferation and/or adominantly Th1-type cytokine response (e.g., IL-2, IFN-γ, and/or TNF-αproduction by T-cells and/or NK cells; and/or IL-12 production bymonocytes, macrophages and/or B cells). Immunogenic portions of theantigens described herein may generally be identified using techniquesknown to those of ordinary skill in the art, including therepresentative methods provided herein.

The compositions and methods of the present invention also encompassvariants of the above polypeptides. A polypeptide “variant,” as usedherein, is a polypeptide that differs from a native protein in one ormore substitutions, deletions, additions and/or insertions, such thatthe immunogenicity of the polypeptide is not substantially diminished.In other words, the ability of a variant to react with antigen-specificantisera may be enhanced or unchanged, relative to the native protein,or may be diminished by less than 50%, and preferably less than 20%,relative to the native protein. Such variants may generally beidentified by modifying one of the above polypeptide sequences andevaluating the reactivity of the modified polypeptide withantigen-specific antibodies or antisera as described herein. Preferredvariants include those in which one or more portions, such as anN-terminal leader sequence or transmembrane domain, have been removed.Other preferred variants include variants in which a small portion(e.g., 1-30 amino acids, preferably 5-15 amino acids) has been removedfrom the N- and/or C-terminal of the mature protein.

Polypeptide variants encompassed by the present invention include thoseexhibiting at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more identity (determined as described below)to the polypeptides disclosed herein.

Preferably, a variant contains conservative substitutions. A“conservative substitution” is one in which an amino acid is substitutedfor another amino acid that has similar properties, such that oneskilled in the art of peptide chemistry would expect the secondarystructure and hydropathic nature of the polypeptide to be substantiallyunchanged. Amino acid substitutions may generally be made on the basisof similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity and/or the amphipathic nature of the residues. Forexample, negatively charged amino acids include aspartic acid andglutamic acid; positively charged amino acids include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values include leucine, isoleucine and valine;glycine and alanine; asparagine and glutamine; and serine, threonine,phenylalanine and tyrosine. Other groups of amino acids that mayrepresent conservative changes include: (1) ala, pro, gly, glu, asp,gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala,phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. A variant may also,or alternatively, contain nonconservative changes. In a preferredembodiment, variant polypeptides differ from a native sequence bysubstitution, deletion or addition of five amino acids or fewer.Variants may also (or alternatively) be modified by, for example, thedeletion or addition of amino acids that have minimal influence on theimmunogenicity, secondary structure and hydropathic nature of thepolypeptide.

Polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes a protein or a portion thereof) or may comprise avariant, or a biological or antigenic functional equivalent of such asequence. Polynucleotide variants may contain one or more substitutions,additions, deletions and/or insertions, as further described below,preferably such that the immunogenicity of the encoded polypeptide isnot diminished, relative to a native tumor protein. The effect on theimmunogenicity of the encoded polypeptide may generally be assessed asdescribed herein. The term “variants” also encompasses homologous genesof xenogenic origin.

When comparing polynucleotide or polypeptide sequences, two sequencesare said to be “identical” if the sequence of nucleotides or amino acidsin the two sequences is the same when aligned for maximumcorrespondence, as described below. Comparisons between two sequencesare typically performed by comparing the sequences over a comparisonwindow to identify and compare local regions of sequence similarity. A“comparison window” as used herein, refers to a segment of at leastabout 20 contiguous positions, usually 30 to about 75, 40 to about 50,in which a sequence may be compared to a reference sequence of the samenumber of contiguous positions after the two sequences are optimallyaligned.

Optimal alignment of sequences for comparison may be conducted using theMegalign program in the Lasergene suite of bioinformatics software(DNASTAR, Inc., Madison, Wis.), using default parameters. This programembodies several alignment schemes described in the followingreferences: Dayhoff, M. O. (1978) A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E. W.and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971) Comb. Theor11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, P.H. A. and Sokal, R. R. (1973) Numerical Taxonomy—the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, W. J. and Lipman, D. J. (1983) Proc. Natl. Acad., Sci. USA80:726-730.

Alternatively, optimal alignment of sequences for comparison may beconducted by the local identity algorithm of Smith and Waterman (1981)Add. APL. Math 2:482, by the identity alignment algorithm of Needlemanand Wunsch (1970) J. Mol. Biol. 48:443, by the search for similaritymethods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT,BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or byinspection.

One preferred example of algorithms that are suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al. (1977)Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol.215:403-410, respectively. BLAST and BLAST 2.0 can be used, for examplewith the parameters described herein, to determine percent sequenceidentity for the polynucleotides and polypeptides of the invention.Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information. In one illustrativeexample, cumulative scores can be calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0) and N (penalty score for mismatching residues;always <0). For amino acid sequences, a scoring matrix can be used tocalculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, andexpectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff andHenikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments, (B) of50, expectation (E) of 10, M=5, N=−4 and a comparison of both strands.

Preferably, the “percentage of sequence identity” is determined bycomparing two optimally aligned sequences over a window of comparison ofat least 20 positions, wherein the portion of the polynucleotide orpolypeptide sequence in the comparison window may comprise additions ordeletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent,or 10 to 12 percent, as compared to the reference sequences (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid bases or amino acidresidue occurs in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the reference sequence (i.e., the window size) andmultiplying the results by 100 to yield the percentage of sequenceidentity.

Therefore, the present invention encompasses polynucleotide andpolypeptide sequences having substantial identity to the sequencesdisclosed herein, for example those comprising at least 50% sequenceidentity, preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99% or higher, sequence identity compared to apolynucleotide or polypeptide sequence of this invention using themethods described herein, (e.g., BLAST analysis using standardparameters, as described below). One skilled in this art will recognizethat these values can be appropriately adjusted to determinecorresponding identity of proteins encoded by two nucleotide sequencesby taking into account codon degeneracy, amino acid similarity, readingframe positioning and the like.

In additional embodiments, the present invention provides isolatedpolynucleotides and polypeptides comprising various lengths ofcontiguous stretches of sequence identical to or complementary to one ormore of the sequences disclosed herein. For example, polynucleotides areprovided by this invention that comprise at least about 15, 20, 30, 40,50, 75, 100, 150, 200, 300, 400, 500 or 1000 or more contiguousnucleotides of one or more of the sequences disclosed herein as well asall intermediate lengths there between. It will be readily understoodthat “intermediate lengths”, in this context, means any length betweenthe quoted values, such as 16, 17, 18, 19, etc.; 21, 22, 23, etc.; 30,31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151,152, 153, etc.; including all integers through 200-500; 500-1,000, andthe like.

The polynucleotides of the present invention, or fragments thereof,regardless of the length of the coding sequence itself, may be combinedwith other DNA sequences, such as promoters, polyadenylation signals,additional restriction enzyme sites, multiple cloning sites, othercoding segments, and the like, such that their overall length may varyconsiderably. It is therefore contemplated that a nucleic acid fragmentof almost any length may be employed, with the total length preferablybeing limited by the ease of preparation and use in the intendedrecombinant DNA protocol. For example, illustrative DNA segments withtotal lengths of about 10,000, about 5000, about 3000, about 2,000,about 1,000, about 500, about 200, about 100, about 50 base pairs inlength, and the like, (including all intermediate lengths) arecontemplated to be useful in many implementations of this invention.

In other embodiments, the present invention is directed topolynucleotides that are capable of hybridizing under moderatelystringent conditions to a polynucleotide sequence provided herein, or afragment thereof, or a complementary sequence thereof. Hybridizationtechniques are well known in the art of molecular biology. For purposesof illustration, suitable moderately stringent conditions for testingthe hybridization of a polynucleotide of this invention with otherpolynucleotides include prewashing in a solution of 5× SSC, 0.5% SDS,1.0 mM EDTA (pH 8.0); hybridizing at 50° C-65° C., 5× SSC, overnight;followed by washing twice at 65° C. for 20 minutes with each of 2×, 0.5×and 0.2× SSC containing 0.1% SDS.

Moreover, it will be appreciated by those of ordinary skill in the artthat, as a result of the degeneracy of the genetic code, there are manynucleotide sequences that encode a polypeptide as described herein. Someof these polynucleotides bear minimal homology to the nucleotidesequence of any native gene. Nonetheless, polynucleotides that vary dueto differences in codon usage are specifically contemplated by thepresent invention. Further, alleles of the genes comprising thepolynucleotide sequences provided herein are within the scope of thepresent invention. Alleles are endogenous genes that are altered as aresult of one or more mutations, such as deletions, additions and/orsubstitutions of nucleotides. The resulting mRNA and protein may, butneed not, have an altered structure or function. Alleles may beidentified using standard techniques (such as hybridization,amplification and/or database sequence comparison).

“Polypeptides” as described herein also include combinationpolypeptides, also referred to as fusion proteins. A “combinationpolypeptide” is a polypeptide comprising at least one of the aboveimmunogenic portions and one or more additional immunogenic Leishmaniasequences, which are joined via a peptide linkage into a single aminoacid chain. The sequences may be joined directly (i.e., with nointervening amino acids) or may be joined by way of a linker sequence(e.g., Gly-Cys-Gly) that does not significantly diminish the immunogenicproperties of the component polypeptides.

Fusion proteins may generally be prepared using standard techniques,including chemical conjugation. Preferably, a fusion protein isexpressed as a recombinant protein, allowing the production of increasedlevels, relative to a non-fused protein, in an expression system.Briefly, DNA sequences encoding the polypeptide components may beassembled separately, and ligated into an appropriate expression vector.The 3′ end of the DNA sequence encoding one polypeptide component isligated, with or without a peptide linker, to the 5′ end of a DNAsequence encoding the second polypeptide component so that the readingframes of the sequences are in frame. This permits translation into asingle fusion protein that retains the biological activity of bothcomponent polypeptides.

A peptide linker sequence may be employed to separate the first and thesecond polypeptide components by a distance sufficient to ensure thateach polypeptide folds into its secondary and tertiary structures. Sucha peptide linker sequence is incorporated into the fusion protein usingstandard techniques well known in the art. Suitable peptide linkersequences may be chosen based on the following factors: (1) theirability to adopt a flexible extended conformation; (2) their inabilityto adopt a secondary structure that could interact with functionalepitopes on the first and second polypeptides; and (3) the lack ofhydrophobic or charged residues that might react with the polypeptidefunctional epitopes. Preferred peptide linker sequences contain Gly, Asnand Ser residues. Other near neutral amino acids, such as Thr and Alamay also be used in the linker sequence. Amino acid sequences which maybe usefully employed as linkers include those disclosed in Maratea etal., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad Sci. USA83:8258-8262, 1986; U.S. Pat. Nos. 4,935,233 and 4,751,180. The linkersequence may generally be from 1 to about 50 amino acids in length.Linker sequences are not required when the first and second polypeptideshave non-essential N-terminal amino acid regions that can be used toseparate the functional domains and prevent steric interference.

The ligated DNA sequences are operably linked to suitabletranscriptional or translational regulatory elements. The regulatoryelements responsible for expression of DNA are located only 5′ to theDNA sequence encoding the first polypeptides. Similarly, stop codonsrequired to end translation and transcription termination signals areonly present 3′ to the DNA sequence encoding the second polypeptide. Thepreparation of fusion proteins of Leishmania antigens is described indetail below in Example 19.

In general, Leishmania antigens having immunogenic properties, and DNAsequences encoding such antigens, may be prepared using any of a varietyof procedures from one or more Leishmania species including, but notlimited to, L. donovani, L. chagasi, L. infantum, L. major, L.amazonensis, L. braziliensis, L. panamensis, L. mexicana, L. tropica,and L. guyanensis. Such species are available, for example, from theAmerican Type Culture Collection (ATCC), Rockville, Md. For example,peptides isolated from MHC class II molecules of macrophages infectedwith a Leishmania species may be used to rescue the correspondingLeishmania donor antigens. MHC class II molecules are expressed mainlyby cells of the immune system, including macrophages. These moleculespresent peptides, which are usually 13-17 amino acids long, derived fromforeign antigens that are degraded in cellular vesicles. The boundpeptide antigens are then recognized by CD4 T-cells. Accordingly,foreign peptides isolated from MHC class II molecules of, for example,Leishmania-infected murine macrophages may be used to identifyimmunogenic Leishmania proteins.

Briefly, peptides derived from Leishmania antigens may be isolated bycomparing the reverse phase HPLC profile of peptides extracted frominfected macrophages with the profile of peptides extracted fromuninfected cells. Peptides giving rise to distinct HPLC peaks unique toinfected macrophages may then be sequenced using, for example, Edmanchemistry as described in Edman and Berg, Eur J Biochem, 80:116-132(1967). A DNA fragment corresponding to a portion of a Leishmania geneencoding the peptide may then be amplified from a Leishmania cDNAlibrary using an oligonucleotide sense primer derived from the peptidesequence and an oligo dT antisense primer. The resulting DNA fragmentmay then be used as a probe to screen a Leishmania library for a fulllength cDNA or genomic clone that encodes the Leishmania antigen. Suchscreens may generally be performed using techniques well known to thoseof ordinary skill in the art, such as those described in Sambrook etal., Molecular Cloning: 4 Laboratory Manual, Cold Spring HarborLaboratories, Cold Spring Harbor, N.Y. (1989).

This approach may be used to identify a 23 kD Leishmania donovaniantigen (referred to herein as Ldp23). The sequence of a polynucleotideencoding Ldp23 is provided in SEQ ID NO:3 and the amino acid sequence ofLdp23 is provided in SEQ ID NO: A Using the methods described herein,Ldp23 has been shown to induce a Th1 immune response in T-cells preparedfrom Leishmania-infected mice.

Alternatively, a Leishmania cDNA or genomic expression library may bescreened with serum from a Leishmania-infected individual, usingtechniques well known to those of ordinary skill in the art.Polynucleotides encoding reactive antigens may then be used to expressthe recombinant antigen for purification. The immunogenic properties ofthe purified Leishmania antigens may then be evaluated using, forexample the representative methods described herein.

For example, sera from Leishmania-infected mice may be used to screen acDNA library prepared from Leishmania amastigotes. Reactive clones maythen be expressed and recombinant proteins assayed for the ability tostimulate T-cells or NK cells derived from Leishmania-immune individuals(i.e., individuals having evidence of infection, as documented bypositive serological reactivity with Leishmania-specific antibodiesand/or a Leishmania-specific DTH response, without clinical symptoms ofleishmaniasis). This procedure may be used to obtain a recombinantpolynucleotide encoding the Leishmania antigen designated M15. Thesequence of such a polynucleotide is provided in SEQ ID NO:1, and theamino acid sequence of the encoded protein is provided in SEQ ID NO:2.

A similar approach may be used to isolate a genomic polynucleotideencoding an immunogenic Leishmania braziliensis antigen, referred toherein as Lbhsp83. More specifically, a genomic clone encoding Lbhsp83may be isolated by screening a L. braziliensis expression library withsera from a Leishmania-infected individual. The DNA encoding Lbhsp83 ishomologous to the gene encoding the eukaryotic 83 kD heat shock protein.The sequence of a polynucleotide encoding nearly all of Lbhsp83 ispresented in SEQ ID NO:5, and the encoded amino acid sequence isprovided in SEQ ID NO:6. Using the methods described below, Lbhsp83 hasbeen found to stimulate proliferation, and a mixed Th1 and Th2 cytokineprofile, in PBMC isolated from L. braziliensis-infected patients.Accordingly, Lbhsp83 is an immunogenic Leishmania antigen. Regions ofLbhsp83 that are not conserved with the mammalian gene have been foundto be particularly potent for T-cell stimulation and antibody binding.Such regions may be identified, for example, by visual inspection of thesequence comparison provided in FIG. 19.

This approach may also be used to isolate a polynucleotide encoding a210 kD immunogenic L. tropica antigen, referred to herein as Lt-210. Thepreparation and characterization of Lt-210, and immunogenic portionsthereof (such as Lt-1 and immunogenic repeat and non-repeat sequences),is described in detail in U.S. patent application Ser. No. 08/511,872,filed Aug. 4, 1995. The sequence of a polynucleotide encoding Lt-1 isprovided in SEQ ID NO:7 and the encoded amino acid sequence is presentedin SEQ ID NO:8.

The above approach may further be used to isolate a polynucleotideencoding a L. braziliensis antigen referred to herein as LbeIF4A.Briefly, such a clone may be isolated by screening a L. braziliensisexpression library with sera obtained from a patient afflicted withmucosal leishmaniasis, and analyzing the reactive antigens for theability to stimulate proliferative responses and preferential Th1cytokine production in PBMC isolated from Leishmania-infected patients,as described below. The preparation and characterization of LbeIF4A isdescribed in detail in U.S. patent application Ser. Nos. 08/454,036 and08/488,386, which are continuations-in-part of U.S. patent applicationSer. No. 08/232,534, filed Apr. 22, 1994. The sequence of apolynucleotide encoding LbeIF4A is provided in SEQ ID NO:9 and theencoded amino acid sequence is presented in SEQ ID NO:10. Homologs ofLbeIF4A, such as that found in L. major, may also be isolated using thisapproach, and are within the scope of the present invention.

Compositions of the present invention may also, or alternatively,contain soluble Leishmania antigens. As used herein, “soluble Leishmaniaantigens” refers to a mixture of at least 8 different Leishmaniaantigens that may be isolated from the supernatant of Leishmaniapromastigotes of any species grown for 8-12 hours in protein-freemedium. Briefly, the organisms are grown to late log phase in complexmedium with serum until they reach a density of 2-3×10⁷ viable organismsper mL of medium. The organisms are thoroughly washed to remove mediumcomponents and resuspended at 2-3×10⁷ viable organisms per mL of definedserum-free medium consisting of equal parts RPMI 1640 and medium 199,both from Gibco BRL, Gaithersburg, Md. After 8-12 hours, the supernatantcontaining soluble Leishmania antigens is removed, concentrated 10 foldand dialyzed against phosphate-buffered saline for 24 hours. Thepresence of at least eight different antigens within the mixture ofLeishmania antigens may be confirmed using SDS-PAGE (i.e., through theobservation of at least 8 different bands). The immunogenic propertiesof the soluble Leishmania antigens may be confirmed by evaluating theability of the preparation to elicit an immune response in cultures oflymph node cells and/or peripheral blood mononuclear cells (PBMC)isolated from presently or previously Leishmania-infected individuals.Such an evaluation may be performed as described below.

Individual antigens present within the mixture of soluble Leishmaniaantigens may be isolated by immunizing mice or rabbits with Leishmaniaculture supernatant, containing soluble antigens, and employing theresultant sera to screen a Leishmania cDNA expression library asdescribed in detail below. This procedure may be used to isolaterecombinant polynucleotides encoding the L. major antigens referred toherein as Lmsp1a, Lmsp9a and MAPS-1A. DNA sequences encoding Lmsp1a,Lmsp9a and MAPS-1A are provided in SEQ ID NO: 19, 21 and 23,respectively, with the corresponding predicted amino acid sequencesbeing presented in SEQ ID NO: 20, 22 and 24, respectively. Similarly,sera from mice or rabbits immunized with L. major culture supernatantmay be used to screen an L. major genomic DNA library. As detailedbelow, this procedure may be used to isolate polynucleotides encodingthe L. major antigens referred to herein as LmgSP1, LmgSP3, LmgSP5,LmgSP8, LmgSP9, LmgSP13, LmgSP19, and polynucleotides encoding the L.chagasi antigens LcgSP1, LcgSP3, LcgSP4, LcgSP8, and LcgSP10. The DNAsequences encoding these antigens are provided in SEQ ID NO:29-35 and44-48, respectively, with the corresponding amino acid sequences beingprovided in SEQ ID NO: 36-42 and 49-53. The L. major antigens referredto herein as 1G6-34, 1E6-44, 4A5-63, 1B11-39, 2A10-37, 4G2-83, 4H6-41and 8G3-100 may be isolated by means of CD4+ T cell expression cloningas described below. DNA sequences encoding these antigens are providedin SEQ ID NO: 72-79, respectively, with the corresponding predictedamino acid sequences being provided in SEQ ID NO: 80-87. The immunogenicproperties of the isolated Leishmania antigens may be evaluated using,for example, the representative methods described herein.

Regardless of the method of preparation, the antigens described hereinare immunogenic. In other words, the antigens (and immunogenic portionsthereof) are capable of eliciting an immune response in cultures oflymph node cells and/or peripheral blood mononuclear cells (PBMC)isolated from presently or previously Leishmania- infected individuals.More specifically, the antigens, and immunogenic portions thereof, havethe ability to induce T-cell proliferation and/or to elicit a dominantlyTh1-type cytokine response (e.g., IL-2, IFN-γ, and/or TNF-α productionby T-cells and/or NK cells; and/or IL-12 production by monocytes,macrophages and/or B cells) in cells isolated from presently orpreviously Leishmania-infected individuals. A Leishmania-infectedindividual may be afflicted with a form of leishmaniasis (such assubclinical, cutaneous, mucosal or active visceral) or may beasymptomatic. Such individuals may be identified using methods known tothose of ordinary skill in the art. Individuals with leishmaniasis maybe identified based on clinical findings associated with at least one ofthe following: isolation of parasite from lesions, a positive skin testwith Leishmania lysate or a positive serological test. Asymptomaticindividuals are infected individuals who have no signs or symptoms ofthe disease. Such individuals can be identified based on a positiveserological test and/or skin test with Leishmania lysate.

The term “PBMC,” which refers to a preparation of nucleated cellsconsisting primarily of lymphocytes and monocytes that are present inperipheral blood, encompasses both mixtures of cells and preparations ofone or more purified cell types. PBMC may be isolated by methods knownto those in the art. For example, PBMC may be isolated by densitycentrifugation through, for example, Ficoll™ (Winthrop Laboratories, NewYork). Lymph node cultures may generally be prepared by immunizingBALB/c mice (e.g., in the rear foot pad) with Leishmania promastigotesemulsified in complete Freünd's adjuvant. The draining lymph nodes maybe excised following immunization and T-cells may be purified in ananti-mouse Ig column to remove the B cells, followed by a passagethrough a Sephadex G10 column to remove the macrophages. Similarly,lymph node cells may be isolated from a human following biopsy orsurgical removal of a lymph node.

The ability of a polypeptide (e.g., a Leishmania antigen or a portion orother variant thereof) to induce a response in PBMC or lymph node cellcultures may be evaluated by contacting the cells with the polypeptideand measuring a suitable response. In general, the amount of polypeptidethat is sufficient for the evaluation of about 2×10⁵ cells ranges fromabout 10 ng to about 100 μg, and preferably is about 1-10 μg. Theincubation of polypeptide with cells is typically performed at 37° C.for about 1-3 days. Following incubation with polypeptide, the cells areassayed for an appropriate response. If the response is a proliferativeresponse, any of a variety of techniques well known to those of ordinaryskill in the art may be employed. For example, the cells may be exposedto a pulse of radioactive thymidine and the incorporation of label intocellular DNA measured. In general, a polypeptide that results in atleast a three fold increase in proliferation above background (i.e., theproliferation observed for cells cultured without polypeptide) isconsidered to be able to induce proliferation.

Alternatively, the response to be measured may be the secretion of oneor more cytokines (such as interferon-γ (IFN-γ), interleukin-4 (IL-4),interleukin-12 (p70 and/or p40), interleukin-2 (IL-2) and/or tumornecrosis factor-α (TNF-α) or the change in the level of mRNA encodingone or more specific cytokines. In particular, the secretion ofinterferon-γ, interleukin-2, tumor necrosis factor-α and/orinterleukin-12 is indicative of a Th1 response, which is responsible forthe protective effect against Leishmania. Assays for any of the abovecytokines may generally be performed using methods known to those ofordinary skill in the art, such as an enzyme-linked immunosorbent assay(ELISA). Suitable antibodies for use in such assays may be obtained froma variety of sources such as Chemicon, Temucula, Calif. and PharMingen,San Diego, Calif., and may generally be used according to themanufacturer's instructions. The level of mRNA encoding one or morespecific cytokines may be evaluated by, for example, amplification bypolymerase chain reaction (PCR). In general, a polypeptide that is ableto induce, in a preparation of about 1-3×10⁵ cells, the production of 30pg/mL of IL-12, IL-4, IFN-γ, TNF-α or IL-12 p40, or 10 pg/mL of IL-12p70, is considered able to stimulate production of a cytokine.

Immunogenic portions of the antigens described herein may be preparedand identified using well known techniques, such as those summarized inPaul, Fundamental Immunology, 3rd ed., 243-247 (Raven Press, 1993) andreferences cited therein. Such techniques include screening polypeptidesderived from the native antigen for immunogenic properties using, forexample, the representative techniques described herein. An immunogenicportion of a polypeptide is a portion that, within such representativeassays, generates an immune response (e.g., proliferation and/orcytokine production) that is substantially similar to that generated bythe full length antigen. In other words, an immunogenic portion of anantigen may generate at least about 25%, and preferably at least about50%, of the response generated by the full length antigen in the model,assays described herein.

Portions and other variants of immunogenic Leishmania antigens may begenerated by synthetic or recombinant means. Synthetic polypeptideshaving fewer than about 100 amino acids, and generally fewer than about50 amino acids, may be generated using techniques well known to those ofordinary skill in the art. For example, such polypeptides may besynthesized using any of the commercially available solid-phasetechniques, such as the Merrifield solid-phase synthesis method, whereamino acids are sequentially added to a growing amino acid chain. SeeMerrifield, J. Am. Chem. Soc. 85:2149-2146, 1963. Equipment forautomated synthesis of polypeptides is commercially available fromsuppliers such as Perkin Elmer/Applied BioSystemsDivision, Foster City,Calif., and may be operated according to the manufacturer'sinstructions.

Recombinant polypeptides containing portions and/or variants of a nativeantigen may be readily prepared from a DNA sequence encoding theantigen. For example, supernatants from suitable host/vector systemswhich secrete recombinant protein into culture media may be firstconcentrated using a commercially available filter. Followingconcentration, the concentrate may be applied to a suitable purificationmatrix such as an affinity matrix or an ion exchange resin. Finally, oneor more reverse phase HPLC steps can be employed to further purify arecombinant protein.

In general, any of a variety of expression vectors known to those ofordinary skill in the art may be employed to express recombinantpolypeptides of this invention. Expression may be achieved in anyappropriate host cell that has been transformed or transfected with anexpression vector containing a polynucleotide that encodes a recombinantpolypeptide. Suitable host cells include prokaryotes, yeast and highereukaryotic cells. Preferably, the host cells employed are E. coli, yeastor a mammalian cell line such as COS or CHO. The DNA sequences expressedin this manner may encode naturally occurring antigens, portions ofnaturally occurring antigens, or other variants thereof. For example,variants of a native antigen may generally be prepared using standardmutagenesis techniques, such as oligonucleotide-directed site-specificmutagenesis, and sections of the DNA sequence may be removed to permitpreparation of truncated polypeptides.

In another aspect, the present invention provides epitope repeatsequences, or antigenic epitopes, of a Leishmania antigen, together withpolypeptides comprising at least two such contiguous antigenic epitopes.As used herein an “epitope” is a portion of an antigen that reacts withsera from Leishmania-infected individuals (i.e. an epitope isspecifically bound by one or more antibodies present in such sera). Asdiscussed above, epitopes of the antigens described in the presentapplication may be generally identified using techniques well known tothose of skill in the art.

In one embodiment, antigenic epitopes of the present invention comprisean amino acid sequence provided in SEQ ID NO:43, 56, 57 or 58. Asdiscussed in more detail below, antigenic epitopes provided herein maybe employed in the diagnosis and treatment of Leishmania infection,either alone or in combination with other Leishmania antigens orantigenic epitopes. Antigenic epitopes and polypeptides comprising suchepitopes may be prepared by synthetic means, as described generallyabove and in detail in Example 15.

In certain aspects of the present invention, described in detail below,the polypeptides, antigenic epitopes, fusion proteins and/or solubleLeishmania antigens of the present invention may be incorporated intopharmaceutical compositions or vaccines. For clarity, the term“polypeptide” will be used when describing specific embodiments of theinventive therapeutic compositions and diagnostic methods. However, itwill be clear to one of skill in the art that the antigenic epitopes andfusion proteins of the present invention may also be employed in suchcompositions and methods.

Pharmaceutical compositions comprise one or more polypeptides, each ofwhich may contain one or more of the above sequences (or variantsthereof), and a physiologically acceptable carrier. Vaccines, alsoreferred to as immunogenic compositions, comprise one or more of theabove polypeptides and an immunostimulant, such as an adjuvant (e.g.,LbeIF4A, interleukin-12 or other cytokines) or a liposome (into whichthe polypeptide is incorporated). Many adjuvants contain a substancedesigned to protect the antigen from rapid catabolism, such as aluminumhydroxide or mineral oil, and a stimulator of immune responses, such aslipid A, Bortadella pertussis or Mycobacterium tuberculosis derivedproteins. Certain adjuvants are commercially available as, for example,Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories,Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway,N.J.); AS-2 (SmithKline Beecham, Philadelphia, Pa.); aluminum salts suchas aluminum hydroxide gel (alum) or aluminum phosphate; salts ofcalcium, iron or zinc; an insoluble suspension of acylated tyrosine;acylated sugars; cationically or anionically derivatizedpolysaccharides; polyphosphazenes; biodegradable microspheres;monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF,interleukin-2, -7, -12, and other like growth factors, may also be usedas adjuvants.

Within certain embodiments of the invention, the adjuvant composition ispreferably one that induces an immune response predominantly of the Th1type. By virtue of its ability to induce an exclusive Th1 immuneresponse, the use of LbeIF4A, and variants thereof, as an adjuvant inthe vaccines of the present invention is particularly preferred. Certainother preferred adjuvants for eliciting a predominantly Th1 -typeresponse include, for example, a combination of monophosphoryl lipid A,preferably 3-de-O-acylated monophosphoryl lipid A, together with analuminum salt. MPL® adjuvants are available from Corixa Corporation(Seattle, WA; see, for example, U.S. Pat. Nos. 4,436,727; 4,877,611;4,866,034 and 4,912,094). CpG-containing oligonucleotides (in which theCpG dinucleotide is unmethylated) also induce a predominantly Th1response. Such oligonucleotides are well known and are described, forexample, in WO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and5,856,462. Immunostimulatory DNA sequences are also described, forexample, by Sato et al., Science 273:352, 1996. Another preferredadjuvant comprises a saponin, such as Quil A, or derivatives thereof,including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham,Mass.); Escin; Digitonin; or Gypsophila or Chenopodium quinoa saponins.Other preferred formulations include more than one saponin in theadjuvant combinations of the present invention, for example combinationsof at least two of the following group comprising QS21, QS7, Quil A,β-escin, or digitonin.

Alternatively the saponin formulations may be combined with vaccinevehicles composed of chitosan or other polycationic polymers,polylactide and polylactide-co-glycolide particles, poly-N-acetylglucosamine-based polymer matrix, particles composed of polysaccharidesor chemically modified polysaccharides, liposomes and lipid-basedparticles, particles composed of glycerol monoesters, etc. The saponinsmay also be formulated in the presence of cholesterol to formparticulate structures such as liposomes or ISCOMs. Furthermore, thesaponins may be formulated together with a polyoxyethylene ether orester, in either a non-particulate solution or suspension, or in aparticulate structure such as a paucilamelar liposome or ISCOM. Thesaponins may also be formulated with excipients such as Carbopol^(R) toincrease viscosity, or may be formulated in a dry powder form with apowder excipient such as lactose.

In one preferred embodiment, the adjuvant system includes thecombination of a monophosphoryl lipid A and a saponin derivative, suchas the combination of QS21 and 3D-MPL® adjuvant, as described in WO94/00153, or a less reactogenic composition where the QS21 is quenchedwith cholesterol, as described in WO 96/33739. Other preferredformulations comprise an oil-in-water emulsion and tocopherol. Anotherparticularly preferred adjuvant formulation employing QS21, 3D-MPL®adjuvant and tocopherol in an oil-in-water emulsion is described in WO95/17210.

Another enhanced adjuvant system involves the combination of aCpG-containing oligonucleotide and a saponin derivative particularly thecombination of CpG and QS21 is disclosed in WO 00/09159. Preferably theformulation additionally comprises an oil in water emulsion andtocopherol.

Additional illustrative adjuvants for use in the compositions of theinvention include Montanide ISA 720 (Seppic, France), SAF (Chiron,Calif., United States), ISCOMS (CSL), MF-59 (Chiron), the SBAS series ofadjuvants (e.g., SBAS-2 or SBAS-4, available from SmithKline Beecham,Rixensart, Belgium), Enhanzyn™ (Corixa, Hamilton, Mont.), RC-529(Corixa, Hamilton, Mont.) and other aminoalkyl glucosaminide4-phosphates (AGPs), such as those described in U.S. Pat. No. 6,113,918and pending U.S. patent application Ser. No. 09/074,720, the disclosuresof which are incorporated herein by reference in their entireties, andpolyoxyethylene ether adjuvants such as those described in WO99/52549A1.

Other preferred adjuvants include adjuvant molecules of the generalformula (i):

HO(CH₂CH₂O)_(n)—A—R,

wherein, n is 1-50, A is a bond or —C(O)—, R is C₁₋₅₀ alkyl or PhenylC₁₋₅₀ alkyl.

One embodiment of the present invention consists of a vaccineformulation comprising a polyoxyethylene ether of general formula (I),wherein n is between 1 and 50, preferably 4-24, most preferably 9; the Rcomponent is C₁₋₅₀, preferably C₄-C₂₀ alkyl and most preferably C₁₂alkyl, and A is a bond. The concentration of the polyoxyethylene ethersshould be in the range 0.1-20%, preferably from 0.1-10%, and mostpreferably in the range 0.1-1%. Preferred polyoxyethylene ethers areselected from the following group: polyoxyethylene-9-lauryl ether,polyoxyethylene-9-steoryl ether, polyoxyethylene-8-steoryl ether,polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, andpolyoxyethylene-23-lauryl ether. Polyoxyethylene ethers such aspolyoxyethylene lauryl ether are described in the Merck index (12^(th)edition: entry 7717). These adjuvant molecules are described in WO99/52549. The polyoxyethylene ether according to the general formula (I)above may, if desired, be combined with another adjuvant. For example, apreferred adjuvant combination is preferably with CpG as described inthe pending UK patent application GB 9820956.2.

Vaccines may additionally contain a delivery vehicle, such as abiodegradable microsphere (disclosed, for example, in U.S. Pat. Nos.4,897,268 and 5,075,109). Pharmaceutical compositions and vaccineswithin the scope of the present invention may also contain otherLeishmania antigens, either incorporated into a combination polypeptideor present within one or more separate polypeptides.

Alternatively, a pharmaceutical or immunogenic composition may containan immunostimulant, such as an adjuvant (e.g., LbeIF4A, interleukin-12or other cytokines, or DNA coding for such enhancers), and DNA encodingone or more of the polypeptides or fusion proteins described above, suchthat the polypeptide is generated in situ. In such compositions, the DNAmay be present within any of a variety of delivery systems known tothose of ordinary skill in the art, including nucleic acid expressionsystems, bacteria and viral expression systems. Appropriate nucleic acidexpression systems contain the necessary DNA sequences for expression inthe patient (such as a suitable promoter and terminating signal).Bacterial delivery systems involve the administration of a bacterium(such as Bacillus-Calmette-Guerrin) that expresses an immunogenicportion of the polypeptide on its cell surface. In a preferredembodiment, the DNA may be introduced using a viral expression system(e.g., vaccinia or other pox virus, retrovirus, or adenovirus), whichmay involve the use of a non-pathogenic (defective), replicationcompetent virus. Techniques for incorporating DNA into such expressionsystems are well known to those of ordinary skill in the art. The DNAmay also be “naked,” as described, for example, in Ulmer et al., Science259:1745-1749 (1993) and reviewed by Cohen, Science 259:1691-1692(1993). The uptake of naked DNA may be increased by coating the DNA ontobiodegradable beads, which are efficiently transported into the cells.

While any suitable carrier known to those of ordinary skill in the artmay be employed in the pharmaceutical compositions of this invention,the type of carrier will vary depending on the mode of administration.For parenteral administration, such as subcutaneous injection, thecarrier preferably comprises water, saline, alcohol, a fat, a wax or abuffer. For oral administration, any of the above carriers or a solidcarrier, such as mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, talcum, cellulose, glucose, sucrose, and magnesiumcarbonate, may be employed. Biodegradable microspheres (e.g., polylacticgalactide) may also be employed as carriers for the pharmaceuticalcompositions of this invention. Suitable biodegradable microspheres aredisclosed, for example, in U.S. Pat. Nos. 4,897,268 and 5,075,109.

In one preferred embodiment, compositions of the present inventioninclude multiple polypeptides selected so as to provide enhancedprotection against a variety of Leishmania species. Such polypeptidesmay be selected based on the species of origin of the native antigen orbased on a high degree of conservation of amino acid sequence amongdifferent species of Leishmania. A combination of individualpolypeptides may be particularly effective as a prophylactic and/ortherapeutic vaccine because (1) stimulation of proliferation and/orcytokine production by a combination of individual polypeptides may beadditive, (2) stimulation of proliferation and/or cytokine production bya combination of individual polypeptides may be synergistic, (3) acombination of individual polypeptides may stimulate cytokine profilesin such a way as to be complementary to each other and/or (4) individualpolypeptides may be complementary to one another when certain of themare expressed more abundantly on the individual species or strain ofLeishmania responsible for infection. A preferred combination containspolypeptides that comprise immunogenic portions of M15, Ldp23, Lbhsp83,Lt-1 and LbeIF4A. Alternatively, or in addition, the combination mayinclude one or more polypeptides comprising immunogenic portions ofother Leishmania antigens disclosed herein, and/or soluble Leishmaniaantigens.

In another preferred embodiment, compositions of the present inventioninclude single polypeptides selected so as to provide enhancedprotection against a variety of Leishmania species. A single individualpolypeptide may be particularly effective as a prophylactic and/ortherapeutic vaccine for those reasons stated above for combinations ofindividual polypeptides.

In another embodiment, compositions of the present invention includeindividual polypeptides and combinations of the above describedpolypeptides employed with a variety of adjuvants, such as IL-12(protein or DNA) to confer a protective response against a variety ofLeishmania species.

In yet another embodiment, compositions of the present invention includeDNA constructs of the various Leishmania species employed alone or incombination with variety of adjuvants, such as IL-12 (protein or DNA) toconfer a protective response against a variety of Leishmania species.

The above pharmaceutical compositions and vaccines may be used, forexample, to induce protective immunity against Leishmania in a patient,such as a human or a dog, to prevent leishmaniasis. Appropriate dosesand methods of administration for this purposes are described in detailbelow.

The pharmaceutical and immunogenic compositions described herein mayalso be used to stimulate an immune response, which may be cellularand/or humoral, in a patient. For Leishmania-infected patients, theimmune responses that may be generated include a preferential Th1 immuneresponse (i.e., a response characterized by the production of thecytokines interleukin-1, interleukin-2, interleukin-12 and/orinterferon-γ, as well as tumor necrosis factor-α). For uninfectedpatients, the immune response may be the production of interleukin-12and/or interleukin-2, or the stimulation of gamma delta T-cells. Ineither category of patient, the response stimulated may include IL-12production. Such responses may also be elicited in biological samples ofPBMC or components thereof derived from Leishmania-infected oruninfected individuals. As noted above, assays for any of the abovecytokines may generally be performed using methods known to those ofordinary skill in the art, such as an enzyme-linked immunosorbent assay(ELISA).

Suitable pharmaceutical compositions and vaccines for use in this aspectof the present invention are those that contain at least one polypeptidecomprising an immunogenic portion of a Leishmania antigen disclosedherein (or a variant thereof). Preferably, the polypeptides employed inthe pharmaceutical compositions and vaccines are complementary, asdescribed above. Soluble Leishmania antigens, with or without additionalpolypeptides, may also be employed.

The pharmaceutical compositions and vaccines described herein may alsobe used to treat a patient afflicted with a disease responsive to IL-12stimulation. The patient may be any warm-blooded animal, such as a humanor a dog. Such diseases include infections (which may be, for example,bacterial, viral or protozoan) or diseases such as cancer. In oneembodiment, the disease is leishmaniasis, and the patient may displayclinical symptoms or may be asymptomatic. In general, the responsivenessof a particular disease to IL-12 stimulation may be determined byevaluating the effect of treatment with a pharmaceutical composition orvaccine of the present invention on clinical correlates of immunity. Forexample, if treatment results in a heightened Th1 response or theconversion of a Th2 to a Th1 profile, with accompanying clinicalimprovement in the treated patient, the disease is responsive to IL-12stimulation. Polypeptide administration may be as described below, ormay extend for a longer period of time, depending on the indication.Preferably, the polypeptides employed in the pharmaceutical compositionsand vaccines are complementary, as described above. A particularlypreferred combination contains polypeptides that comprise immunogenicportions of M15, Ldp23, Lbhsp83, Lt-1and LbeIF4A, Lmspla, Lmsp9a, andMAPS-1A. Soluble Leishmania antigens, with or without additionalpolypeptides, may also be employed.

Routes and frequency of administration, as well as dosage, for the aboveaspects of the present invention will vary from individual to individualand may parallel those currently being used in immunization againstother infections, including protozoan, viral and bacterial infections.In general, the pharmaceutical compositions and vaccines may beadministered by injection (e.g., intracutaneous, intramuscular,intravenous or subcutaneous), intranasally (e.g., by aspiration) ororally. Between 1 and 12 doses may be administered over a 1 year period.For therapeutic vaccination (i.e., treatment of an infected individual),12 doses are preferably administered, at one month intervals. Forprophylactic use, 3 doses are preferably administered, at 3 monthintervals. In either case, booster vaccinations may be givenperiodically thereafter. Alternate protocols may be appropriate forindividual patients. A suitable dose is an amount of polypeptide or DNAthat, when administered as described above, is capable of raising animmune response in an immunized patient sufficient to protect thepatient from leishmaniasis for at least 1-2 years. In general, theamount of polypeptide present in a dose (or produced in situ by the DNAin a dose) ranges from about 100 ng to about 1 mg per kg of host,typically from about 10 μg to about 100 μg. Suitable dose sizes willvary with the size of the patient, but will typically range from about0.1 mL to about 5 mL.

In another aspect, this invention provides methods for using one or moreof the polypeptides described above to diagnose Leishmania infection ina patient using a skin test. As used herein, a “skin test” is any assayperformed directly on a patient in which a delayed-type hypersensitivity(DTH) reaction (such as induration and accompanying redness) is measuredfollowing intradermal injection of one or more polypeptides as describedabove. Such injection may be achieved using any suitable devicesufficient to contact the polypeptide or polypeptides with dermal cellsof the patient, such as a tuberculin syringe or 1 mL syringe.Preferably, the reaction is measured at least 48 hours after injection,more preferably 72 hours after injection.

The DTH reaction is a cell-mediated immune response, which is greater inpatients that have been exposed previously to a test antigen (i.e., animmunogenic portion of a polypeptide employed, or a variant thereof).The response may measured visually, using a ruler. In general,induration that is greater than about 0.5 cm in diameter, preferablygreater than about 1.0 cm in diameter, is a positive response,indicative of Leishmania infection, which may or may not be manifestedas an active disease.

The polypeptides of this invention are preferably formulated, for use ina skin test, as pharmaceutical compositions containing at least onepolypeptide and a physiologically acceptable carrier, as describedabove. Such compositions typically contain one or more of the abovepolypeptides in an amount ranging from about 1 μg to 100 μg, preferablyfrom about 10 μg to 50 μg in a volume of 0.1 mL. Preferably, the carrieremployed in such pharmaceutical compositions is a saline solution withappropriate preservatives, such as phenol and/or Tween 80™.

The inventive polypeptides may also be employed in combination with oneor more known Leishmania antigens in the diagnosis of leishmaniasis,using, for example, the skin test described above. Preferably,individual polypeptides are chosen in such a way as to be complementaryto each other. Examples of known Leishmania antigens which may beusefully employed in conjunction with the inventive polypeptides includeK39 (Burns et al., Proc. Natl. Acad. Sci. USA, 1993 90:775-779).

The following Examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 Preparation of M15

This Example illustrates the preparation of a Leishmania antigen M15,having the sequence provided in SEQ ID NO:2.

An L. major (Friedlan strain) amastigote cDNA expression libraryprepared in the λZAP II vector (Stratagene, La Jolla, Calif.) wasscreened according to manufacturer's instructions using sera obtainedfrom L. major infected BALB/c mice (8 weeks post inoculation).Approximately 40,000 plaques were screened and four clones expressingreactive antigens were purified to homogeneity by two subsequent roundsof low density screening. Bluescript phagemid inserts were excised frompositive clones for further analysis. An EcoRI/SstII restrictionfragment from the 5′ end of one partial cDNA insert isolated duringfirst round screening (pLmal-1) was subsequently used as a probe torescreen for clones containing full length cDNA inserts. The probe waslabeled to high specific activity (˜10⁹ cpm/μg) with [α-³²P]dCTP usingthe random primer method and was used to screen ˜10,000 plaques of theL. major expression library described above. Positive clones werecompared by restriction enzyme digestion and the clone with the largestinsert (pfl1-1) was chosen for subsequent analysis.

DNA sequence analyses were performed on an Applied Biosystems automatedsequencer using Taq polymerase and dye coupled ddNTP terminators ordye-labeled sequencing primers. The complete sequence of the 2685 bpinsert was determined using a combination of primer-directed sequencingand by sequencing a series of overlapping Exonuclease III deletionsubclones generated using the Erase-a-base system (Promega, Madison,Wis.). The sequence of this insert is provided in SEQ ID NO: 1, and thededuced amino acid sequence is provided in SEQ ID NO:2.

The complete insert of clone pf1-1 was excised by digestion withBamHI/KpnI and was subcloned in frame into BamHI/KpnI digested pQE31(QUIAGEN) to generate the construct pM151A. E. coli containing thisconstruct inducibly expressed high levels of the L. major antigenencoded by pfl1-1 (designated as M15) with the addition of a 6-histidinetag at the amino terminus. Large volume cultures (500 ml) of E. colihost cells containing the pM151 A construct were induced to expressrecombinant protein by the addition of 2 mM IPTG at mid-log phase ofgrowth. Growth was continued for 4 to 5 hours and bacteria were thenpelleted and washed once with cold PBS. Bacteria were resuspended in 20ml of lysis buffer (50 mM Na₂HPO₄, pH 8.0, 300 mM NaCl, 10 mMβ-mercaptoethanol) containing 20 mg of lysozyme and were lysed by a 1hour incubation at 4 ° C. followed by brief sonication. Insolublematerial was removed by centrifugation at 10,000× g for 10 minutes andalthough the recombinant protein was found to be evenly distributedbetween the soluble and insoluble fractions the insoluble material wasdiscarded at this point. Recombinant protein containing the aminoterminal histidine tag was affinity purified using Ni-NTA resin (QIAGEN)according to the manufacturer's recommendations. Briefly, 8 ml of Ni-NTAresin resuspended in lysis buffer was added to the soluble lysatefraction and binding was conducted with constant mixing for 1 hour at 4°C. The mixture was then loaded into a gravity flow column and thenon-binding material was allowed to flow through. The Ni-NTA matrix waswashed 3 times with 25 ml of wash buffer (50 mM Na₂HPO₄, pH 6.0, 300 mMNaCl, 10 mM β-mercaptoethanol) and bound material was eluted in 25 ml ofelution buffer (50 mM Na₂HPO₄, pH 5.0, 300 mM NaCl, 10 mMβ-mercaptoethanol). The eluted material was then dialyzed against 3changes of PBS, sterile filtered and stored at −20° C. The purifiedrecombinant protein was shown by SDS-PAGE analysis to be free of anysignificant amount of E. coli protein. A small number of bands of lowermolecular weight were assumed to be proteolytic products of the L. majorantigen based on their reactivity by western blot analysis. A high titrepolyclonal antisera against M15 was generated in rabbits by repeatedsubcutaneous injection of recombinant protein. Western blot analysis oflysates from L. major promastigotes and amastigotes using this antiseraindicated that the protein is constitutively expressed throughout theparasite lifecycle.

Example 2 Preparation of LDP23

This Example illustrates the preparation of a Leishmania antigen Ldp23,having the sequence provided in SEQ ID NO:4.

A. Purification of MHC Class II-associated Peptides from P388D1Macrophages Infected with L. donovani

To ascertain that in vitro infection of macrophages would load their MHCclass II molecules with parasite peptides, initial experiments werecarried out to test the ability of L. donovani-infected macrophage cellline P388D1 to present parasite antigens to L. donovani specificT-cells. This macrophage cell line was chosen because it has the sameH-2 haplotype as the BALB/c mouse, which is a strain of mouse moderatelysusceptible to L. donovani infection and selected to conduct the in vivoexperiments. Using a proportion of 3-5 parasites per cell and an initialincubation at room temperature for 4-6 hours follows by 37° C. for 24-48hours, close to 90% of the macrophages were infected. The level of MHCclass II molecule expression, as determined by FACS analysis, indicatedthat infection did not cause an effect on the levels of MHC class IIexpression when compared to non-infected control cells.

To test the ability of the L. donovani-infected P388D1 cells to presentparasite antigens, macrophages were infected as indicated above andincubated at 26° C. for 6 hours, and then as 37° C. for either 24, 48 or72 hours. At each of these time points the non-adherent cells and freeparasites were washed out and the adherent cells were mechanicallydislodged, washed and fixed with paraformaldehyde. These cells were thenused as antigen presenting cells (APCs) for purified lymph node T-cellsfrom BALB/c mice immunized with L. donovani promastigotes. To generatethese anti-L. donovani specific T-cells, BALB/c mice (H-2^(d)) of bothsexes (The Jackson Laboratory, Bar Harbor, Me.) were immunized at 8 to14 weeks of age in the rear foot pad with 5-10×10⁶ L. donovanipromastigotes emulsified in complete Freund's adjuvant (CFA) (DifcoLaboratories, Madison, Mich.) as described in Rodrigues et al., ParasiteImmunol. 14:49 (1992). The draining lymph nodes were excised 8 daysafter the immunization and T-cells were purified in an anti-mouse Igcolumn to remove the B cells, as described in Bunn-Moreno andCampos-Neto, J. Immunol. 127:427 (1981), followed by a passage through aSephadex G10 column to remove the macrophages.

Stimulation index was calculated by dividing the cpm obtained for thecells cultured in the presence of infected P388D1 macrophages by the cpmobtained for the cells cultured in the presence of non-infectedmacrophages, but subjected to the same conditions as the infectedmacrophages. The results shown FIG. 1 indicate that L. donovani-infectedP388D1 macrophage process parasite antigens and that optimalpresentation occurs after 48 hours of infection. No stimulation of theT-cells by the non-infected macrophages was observed.

To isolate the MHC class II associated L. donovani peptides, P388D1macrophages were infected with L. donovani promastigotes for an initialincubation of 6 hours at room temperature. The cultures were thentransferred to 37° C. for the remainder of the 48 hour incubationperiod. At a ratio of 3-5 parasites per macrophage nearly 90% of themacrophages were infected after 24 hours of incubation at 37° C.

The MHC class II molecules were then affinity-purified. Approximately1.5×10¹⁰ L. donovani-infected or an equal number of non-infected P388D1macrophages were used for each purification. The cells were harvested,washed with PBS and incubated for 30 minutes in cold lysis buffer (PBS,1% Nonidet P40, 25 mM iodoacetamide, 0.04% sodium azide, 1 mM aprotininand 1 mM PMSF). The insoluble material was removed by centrifugation at40,000 g for 1 hour and the supernatant was recycled overnight at 4° C.over a 5 ml anti-MHC class II molecules (H-2d) Sepharose column (ProteinG Sepharose column to which the monoclonal antibody MK-D6 has beenbound). Culture supernatants of MK-D6 hybridoma cells (American TypeCulture Collection, Rockville, Md.) were employed as the source foranti-MHC class II (H-2 d) monoclonal antibody. The column was washedwith 50 ml of lysis buffer and then with 50 ml of PBS containing 0.5%octyl glucopyranoside detergent. Bound molecules were eluted from thecolumn with 1M acetic acid in 0.2% NaCl. The MHC/peptide molecules wereseparated from the IgG (MK-D6 monoclonal antibody) using a Centricon 100filter unit (Amicon Division, W.R. Grace & Co., Beverly, Mass.). Thepeptides were then dissociated from the class II molecules by theaddition of acetic acid to 2.5M, followed by separation using aCentricon 10 filter unit. The resulting peptide preparation, present inthe low molecular weight sample, was then dried using a speed vacconcentrator (Savant Instrument Inc., Farmingdale, N.Y.).

The peptides were redissolved in 200 μl of 0.05% TFA and separated byreverse-phase high performance liquid chromatography (RP-HPLC) using a2.1 mm×25 cm Vydac C-18 column at a flow rate of 0.15 ml/min employing a1 to 30% acetonitrile gradient (60 min) followed by a 30 to 60% gradient(30 min) and then a 60 to 80% gradient (90-110 min). Non-infected P388D1cells were similarly processed to serve as background control forendogenous MHC class II associated peptides. FIG. 2 shows arepresentative experiment; four distinct peaks which are present only inthe material isolated from infected macrophages (panel B), and not inthe material isolated from uninfected macrophages (panel A) areindicated.

Out of three independent peptide extractions, twenty five distinct HPLCpeptide peaks were isolated from L. donovani-infected macrophages andwere subjected to protein sequence analysis using automated Edmandegradation on an Applied Biosystems 477 gas-phase protein sequencer.Protein sequence and amino acid analysis were performed by the W.M. KeckFoundation, Biotechnology Resource Laboratory, Yale University, NewHaven, Conn. In practically all determinations, no assignment could bemade for the first position. Also, in most cases the definition of theamino acid residues of the 10-15 positions was based on the quantitativedominance of one residue over others. Using this approach, the sequencesobtained for several peptides showed the presence of 3-6 differentresidues in many of the 10-15 sequence cycles analyzed for eachdetermination, reflecting a mixture of peptides. In addition, sequencescould not be obtained for some peaks because the peptides were blocked.Notwithstanding, three peptides sequences were determined. Amino-acidsequences were searched for identity with proteins in the GenBankdatabase using the GENPETP, PIR and SWISSPROT programs. The sequencedata base analysis revealed that one of the peptides was highlyhomologous to glyceraldehyde-3-phosphate dehydrogenase of variousspecies. Another peptide had homology with elongation factor of severalspecies, including Leishmania. The third sequence was not clearlyrelated to any known proteins, and is shown below:

XQXPQ(L/K)VFDEXX (SEQ ID NO:11).

B. Cloning and Sequencing of the Ldp23 Gene

In order to retrieve the L. donovani protein that was processed into apeptide associated with the MHC class II molecules of infectedmacrophages, the peptide sequence of uncertain origin was chosen toguide the strategy for cloning the corresponding parasite gene. A DNAfragment was initially amplified from L. donovani promastigote cDNA byPCR. The sense primer was a peptide derived oligonucleotide(5′>GGAATTCCCCInCAGCTInGTInTTCGAC<3′) (SEQ ID NO:12) containing an EcoRIrestriction endonuclease site (underlined). The bases were selectedfollowing the preferential codon usage of L. donovani, as described inLangford et al., Exp. Parasitol. 74:360 (1992). Inosine was used for theresidues of positions 4, 6 and 7 because of the low codon usageassurance for the corresponding amino acids. In addition, thecarboxyl-terminal L-glutamic acid was not included for the design of theprimer. The antisense primer was a poly-thymidine oligonucleotide (oligodT, downstream primer) containing a XhoI restriction endonuclease site.

The gene fragment was amplified from a L. donovani promastigote cDNApreparation using the following reaction conditions: one cycle of 3 minat 94° C. immediately followed by 35 cycles of 1 min at 94° C., 1 min at45° C. and 1 min at 72° C. The L. donovani cDNA was prepared from 5×10⁷washed promastigote forms harvested at the log growth phase (3 daysculture). The cDNA was obtained using an Invitrogen cDNA cycle™ kit(Invitrogen Co., San Diego, Calif.). Oligonucleotide primers weresynthesized by the DNA Synthesis Laboratory, Department of Pathology,Yale University School of Medicine.

The PCR products were analyzed by gel electrophoresis. Only one band ofapproximately 300 bp was obtained. This fragment was cloned and itssequence confirmed the sequence of the peptide-based primer includingthe glutamic acid codon, deliberately not included in the primersequence.

The PCR amplified gene fragment was ligated into the pCR™ vector usingthe TA cloning system (Invitrogen Co., San Diego, Calif.). Transformantswere selected in LB medium containing 100μg/ml ampicillin and theplasmid DNA was isolated using the Wizard™ Minipreps DNA purificationkit (Promega Co., Madison, Wis.). Insert DNA was released with therestriction enzymes EcoRI and XhoI (New England Biolabs, Beverly,Mass.), purified from an agarose gel electrophoresis and labeled with³²P using a random priming method (Megaprime Labeling Kit, Amersham LifeScience, Buckinghamshire, England).

This DNA fragment was used as probe to screen a L. donovani promastigotecDNA library as described in Skeiky et al., Infect. Immun. 62:1643(1994). An approximately 650 bp cDNA (Ldp23) was excised from thephagemid by in vivo excision using the Stratagene protocol. DNAsequencing was performed using the Sequenase version 2 system (DNAsequencing kit) in the presence or absence of 7-deaza-GTP (United StatesBiochemical, Cleveland, Ohio). The sequence is provided as SEQ ID NO:3,and shows complete homology with the original 300 bp PCR fragment. A 525bp open reading frame containing an ATG codon that follows the last 4bases of the spliced leader sequence and 3 stop codons adjacent to thepoly A tail was identified. This frame also codes the carboxyl terminalsequence (KVFDE) (SEQ ID NO:13) of the purified MHC class II associatedpeptide. The sequence analysis of the deduced protein sequence revealedone potential glycosylation site (Asn-Cys-Ser) at positions 68-70.

Sequence analysis was performed using the University of WisconsinGenetics Computer Group Programs and the GenBank and EMBL data bases ofprotein and DNA sequences. The search for homology of the Ldp23 genewith known sequences revealed no significant homology.

C. Bacterial Expression and Purification of Recombinant Protein

The recombinant L. donovani peptide donor protein was produced in E.coli transformed with the pGEX 2T expression vector in which the Ldp23gene was subcloned in frame. PCR was used to subclone the cloned gene inframe into the expression vector pGEX 2T. Primers containing theappropriate restriction site enzymes, initiation and termination codonswere: 5′>GGATCCATGGTCAAGTCCCACTACATCTGC<3′ (SEQ ID NO:14) for theupstream primer and 5′>GAATTCAGACCGGATAGAAATAAGCCAATGAAA<3′ (SEQ IDNO:15) for the downstream primer (restriction sites of BamHI and EcoRIare underlined respectively). PCR conditions were as indicated above forthe amplification of the original peptide related DNA fragment. Thetemplate used was pBluescript plasmid containing the cloned gene fromthe cDNA library.

Overexpression of the recombinant fusion protein was accomplished bygrowing the transformed E. coli (DH5α) and inducing the tac promoterwith 1 mM isopropyl-β-thiogalactopyranoside (IPTG) (Stratagene, LaJolla, Calif.). Cells were collected, centrifuged, and analyzed for thepresence of the fusion protein by SDS-PAGE. A glutathione-S-transferasefusion protein of 43-44 kD was produced, indicating a leishmanialprotein of approximately 18 kD, as glutathione-S-transferase (GST) has aMW of 26 kD. However, the fusion protein was very insoluble andtherefore could not be purified by affinity chromatography using aglutathione column. The use of low concentrations of detergents likeSDS, sarcosyl, deoxycolate, and octyl-glucopyranoside during theextraction steps was efficient to solubilize the protein butunfortunately prevented its binding to the glutathione column. Othermaneuvers, such as the growth of the E. coli and incubation andinduction of the tac promoter with IPTG at 33° C., did not improve theprotein solubility. However, the purification was achieved bypreparative SDS-PAGE. The band was visualized with 0.1M KCl, cut andelectroeluted from the gel followed by extensive dialysis against PBSand concentration on Centricon 10 filters.

Approximately 500 μg of purified protein was obtained. The purifiedprotein is shown in FIG. 3. In panel A, E. coli (DH5α) transformed withthe expression vector pGEX 2T containing the Ldp23 gene was grown in LBmedium and the tac promoter was induced with IPTG for 3 hours. The cellswere pelleted, resuspended in loading buffer and submitted to SDS-PAGE(10%) under reducing condition. The gel was stained with Coomassie blue.Lane 1 shows the uninduced E. coli and land 2 shows the induced E. coli.The arrow indicates the recombinant protein. Panel B shows the proteinprepared as in panel A and submitted to a preparative SDS-PAGE. The bandcorresponding to the overexpressed recombinant fusion protein wasidentified by KCl, cut out, electroeluted from the gel strip, dialyzedagainst PBS and submitted to analytical SDS-PAGE (12%). Numbers on theleft side indicate the molecular weights of the markers. Attempts tofurther purify the leishmanial protein by cleaving it out from thefusion protein GST with thrombin were unsuccessful.

D. Expression of Ldp23

To ascertain that the Ldp23 peptide is expressed in Leishmaniaorganisms, a Northern blot analysis was performed using RNA preparedfrom different promastigote growth phases (logarithmic and stationary)and from the amastigote form of these parasites.

The RNA was prepared from 2×10⁷ parasite cells using the Micro RNAisolation kit (Stratagene, La Jolla, Calif.) according to the company'srecommended instructions. RNA was prepared from L. donovanipromastigotes (logarithmic growth phase); from L. major promastigotes(logarithmic and stationary growth phases); from L. amazonensis, bothpromastigotes (logarithmic and stationary growth phases) and amastigotespurified from CBA/J infected mice; and from L. pifanoi, bothpromastigotes (logarithmic and stationary growth phases) and amastigotes(from axenic culture medium). L. donovani (1S strain), L. amazonensis(MHOM/BR/77/LTB0016), L. major (MHOM/IR/79/LRC-L251) and L. pifanoi(MHOM/VE/60/Ltrod) promastigotes were grown and maintained at 26° C. inSchneider's medium containing 20% FCS and 50 μg/ml gentamicin. Theamastigote forms of L. amazonensis were obtained by differentialcentrifugation of a “pus-like” foot pad lesion of a CBA/J mouse infectedfor 6 months with this parasite. L. pifanoi amastigotes were obtainedfrom axenic culture as previously reported by Pan et al., J. Euk.Microbiol. 40:213 (1993).

The hybridization was carried out at 45° C. in the presence of 50%formamide, 5× Denhardt's solution, 0.1% SDS, 100 μg/ml single strandedsalmon sperm DNA and 5× SSPE using 0.45 μm Nytran membrane filters(Schleicher & Schuell, Keene, NH). The probe was the ³²P labeled Ldp23gene.

FIG. 4 shows that one single RNA band of 680 bp was observed for allgrowth phases and forms of all tested Leishmania. Within FIG. 4, thenumbers 1, 2 and 3 refer to RNA obtained from promastigotes at thelogarithmic growth phase, promastigotes at the stationary growth phaseand amastigote forms, respectively, and the numbers on the left sideindicate the molecular weights of the markers in base pairs. This resultis consistent with the corresponding gene size (525 bp) and with themolecular weight of the expressed protein and points to the ubiquitousdistribution and expression of this gene within the genus Leishmania.

E. Induction of Anti-L. donovani Antibody Response in Mice and Rabbitsby Purified Recombinant Protein

In order to evaluate the immunogenicity of the recombinant leishmanialprotein, and to investigate its expression in the parasites, mice andrabbits were immunized with the GST-fusion protein in CFA. BALB/c micewere immunized in the rear foot pad with 5-10 μg of protein emulsifiedin CFA. Protein concentration was determined using the Bio-Rad ProteinAssay reagent (Bio-Rad Laboratories, Richmond, Calif.). The mice wereboosted 7 days later with 5-10 μg of protein emulsified in incompleteFreund's adjuvant (IFA) inoculated into the peritoneal cavity. The micewere bled 7 days after the second immunization. New Zealand whiterabbits (Millbrook Farm, Amherst, Mass.) were immunized according to thefollowing protocol: one intramuscular (IM) injection of 25-30 μg ofpurified recombinant protein emulsified in CFA into each thigh on dayone; one 1M injection of 25-30 μg of purified protein emulsified in IFAinto each shoulder on day 7; on day 15, 25-30 μg of the purified proteinin PBS was injected into the subcutaneous tissue. The rabbit was bled 7days after the last immunization.

Sera were prepared and the anti-Leishmania antibody response wasmeasured by Western blot analysis and by FACScan. In both cases L.donovani promastigotes were used as antigen. Approximately 2×10⁶ L.donovani promastigotes were grown in Schneider's medium for 3 days (logphase), were washed with PBS, lysed with SDS-PAGE loading buffer andsubmitted to electrophoresis under reducing conditions using a 15%polyacrylamide gel. The proteins were transferred onto 0.45μ Immobilon-Ptransfer membrane (Millipore Co., Bedford, Mass.) using a wet-typeelectroblotter (Mini Trans-Blot Electrophoretic Transfer Cell, Bio RadLife Science Division, Richmond, Calif.) for 2 hours at 50 V. Themembranes were blocked overnight at room temperature with PBS containing3% normal goat serum (NGS), 0.2% Tween-20 and 0.05% sodium azide,followed by 3 washes with PBS. The blots were then incubated for 3-4hours at 4° C. with a {fraction (1/200)} dilution of pre-immune rabbitserum (lane A, FIG. 5) or with the same dilution of anti-fusion proteinrabbit antiserum (lane B, FIG. 5). The sera was previously absorbed 2×with non-viable desiccated Mycobacterium tuberculosis H-37 RA (DifcoLaboratories, Detroit, Mich.) and were diluted in PBS containing 1% NGSand 5% powdered non-fat bovine milk (Carnation, Nestlé Food Company,Glendale, Calif.). The membranes were then washed with PBS, incubatedfor 1 hour at room temperature with goat anti-rabbit IgG antibodyconjugated with alkaline phosphatase (Promega, Madison, Wis.), washedonce with PBS and 2× with veronal buffer pH 9.4. The reaction wasvisualized using the substrate mixture5-bromo-4-chloro-3-indoyl-phosphate and nitroblue tetrazolium(Kirkegaard & Perry Laboratories Inc., Gaithersburg, Md.) according tothe manufacturer's instructions.

FIG. 5 shows that the rabbit anti-recombinant protein antiserum detectsa single protein of 23 kDa (Ldp23) in the Leishmania crude extractantigen preparation. No bands were observed when an anti-GST antiserumwas used (not shown). Moreover, the FACScan analysis (FIG. 6) shows thatthe antibody induced by the recombinant Ldp23 reacts with intact live L.donovani promastigotes, thus pointing to a cell surface expression ofthis molecule on these organisms. The dotted line in FIG. 6 shows theindirect immunofluorescence performed using pre-immune mouse serum andthe solid line in FIG. 6 shows the result obtained with mouseanti-GST-Ldp23 antiserum. Both sera were diluted at {fraction (1/100)}.Parasites were washed with staining buffer and incubated with FITCconjugated goat anti-mouse immunoglobulin antibody. Fluorescenceintensity was analyzed by FACScan.

F. Recognition of Recombinant Ldp23 by Leishmania-specific Lymph NodeT-cells

To test the responsiveness of T-cells to the Ldp23 protein, two sets ofexperiments were performed. In the first experiment, lymph node T-cells(10⁵/well) from BALB/c mice immunized with L. donovani promastigotes (asdescribed above) were stimulated to proliferate with 2×10⁵ MitomycinC-treated normal mononuclear spleen cells (APC) and pulsed with thepurified recombinant fusion protein. Proliferation of T-cells wasmeasured at 72 hours of culture. Values are expressed in FIG. 7 as cpmand represent the mean of [³H]TdR incorporation of triplicate cultures.Background cpm of cells (T cells+APC) cultured in the presence of mediumalone was 1291. FIG. 7 shows that Leishmania specific T-cellsproliferate well and in a dose response manner to recombinant Ldp23. Noresponse was observed when purified GST was added instead of therecombinant fusion protein nor when lymph node T-cells from miceimmunized with CFA alone were stimulated to proliferate in the presenceof the Leishmanial fusion protein (not shown).

The recognition of the recombinant Ldp23 protein by Leishmania-specificT-cells was also tested using two murine models of leishmaniasis, the L.major highly susceptible BALB/c mice and the L. amazonensis susceptibleCBA/J mice as described in Champsi and McMahon-Pratt, Infect. Immun.56:3272 (1988). These models were selected to investigate the cytokinepattern induced by Ldp23. In the mouse model of leishmaniasis,resistance is associated with Th 1 cytokines while susceptibility islinked to Th 2 responses.

Lymph node cells were obtained 3 weeks after the initiation of infectionof BALB/c mice with L. major and the ability of these cells to recognizethe recombinant Ldp23 was measured by proliferation and by theproduction of the cytokines IFN-γ and IL-4. 2×10⁶ cells obtained fromthe draining popliteal lymph node of infected mice were cultured for 72hours in the presence of recombinant Ldp23 or Leishmania lysate. Thelevels of IFN-γ and IL-4 in culture supernatants were measured by ELISAas previously described (Chatelain et al., J. Immunol. 148:1172 (1992),Curry et al., J. Immunol. Meth. 104:137 (1987), and Mossman and Fong, J.Immunol. Meth. 116:151 (1989)) using specific anti IFN-γ and IL-4monoclonal antibodies (PharMingen, San Diego, Calif.).

Ldp23 did stimulate these cells to proliferate (not shown) and induced atypical Th 1 type of cytokine response as indicated by the production ofhigh levels of IFN-γ (panel A of FIG. 8) and no IL-4 (panel B of FIG.8). Stimulation of these cells with a Leishmania crude lysate yielded amixed Th cytokine profile. Exactly the same pattern of cytokineproduction was obtained from the CBA/J mice infected with L. amazonensis(not shown). These results clearly indicate that Ldp23 is a powerful andselective activator of the Th 1 cytokines by mouse cells.

Example 3 Preparation of HsP83

This Example illustrates the preparation of a Leishmania antigen Hsp83,having the sequence provided in SEQ ID NO:6.

A genomic expression library was constructed with sheared DNA from L.braziliensis (MHOM/BR/75/M2903) in bacteriophage λZAP II (Stratagene, LaJolla, Calif.). The expression library was screened with Escherichiacoli preadsorbed serum from an L. braziliensis-infected individual withML. Immunoreactive plaques were purified, and the pBSK(−) phagemid wasexcised by protocols suggested by the manufacturer. Nested deletionswere performed with exonuclease III to generate overlapping deletionsfor single-stranded template preparations and sequencing.Single-stranded templates were isolated following infection with VCSM13helper phage as recommended by the manufacturer (Stratagene, La Jolla,Calif.) and sequenced by the dideoxy chain terminator method or by theTaq dye terminator system using the Applied Biosystems automatedsequencer model 373A.

Recombinant antigens produced by these clones were purified from 500 mlof isopropyl-β-D-thiogalactopyranoside (IPTG)-induced cultures asdescribed in Skeiky et al., J. Exp. Med. 176:201-211 (1992). Theseantigens were then assayed for the ability to stimulate PBMC fromLeishmania-infected individuals to proliferate and secrete cytokine.Peripheral blood was obtained from individuals living in an area (Cortede Pedra, Bahia, Brazil) where L. braziliensis is endemic and whereepidemiological, clinical, and immunological studies have been performedfor over a decade, and PBMC were isolated from whole blood by densitycentrifugation through Ficoll (Winthrop Laboratories, New York, N.Y.).For in vitro proliferation assays, 2×10⁵ to 4×10⁵ cells per well werecultured in complete medium (RPMI 1640 supplemented with gentamicin,2-mercaptoethanol, L-glutamine, and 10% screened pooled A+ human serum;Trimar, Hollywood, Calif.) in 96-well flat-bottom plates with or without10 μg of the indicated antigens per ml or 5 μg of phytohemagglutinin perml (Sigma Immunochemicals, St. Louis, Mo.) for 5 days. The cells werethen pulsed with 1 μCi of [³H]thymidine for the final 18 h of culture.For determination of cytokine production 0.5 to 1 ml of PBMC wascultured at 1×10⁶ to 2×10⁶ cells per ml with or without the Leishmaniaantigens for 48 and 72 h.

The supernatants and cells were harvested and analyzed for secretedcytokine or cytokine mRNAs. Aliquots of the supernatants were assayedfor gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α),interleukin-4 (IL-4), and IL-10 as described in Skeiky et al., J. Exp.Med. 181:1527-1537 (1995). For cytokine mRNA PCR analysis, total RNA wasisolated from PBMC and cDNA was synthesized by using poly(dT)(Pharmacia, Piscataway, N.J.) and avian mycloblastosis virus reversetranscriptase. Following normalization to β-actin, diluted cDNA wasamplified by PCR using Taq polymerase (Perkin-Elmer Cetus, Foster City,Calif.) with 0.2 μM concentrations of the respective 5′ and 3′ externalprimers in a reaction volume of 50 μl. The nucleotide sequences of theprimary pairs and the PCR conditions used were as described in Skeiky etal., J. Exp. Med. 181:1527-1537 (1995). We verified that our PCRconditions were within the semiquantitative range by initiallyperforming serial dilutions of the cDNAs and varying the number ofcycles used for PCR. Plasmids containing the human sequences for IL-2,IFN-γ, IL-4, IL-10, and β-actin were digested, and the DNA inserts werepurified after separation on 1% agarose gels. Radiolabeled ³²P probeswere prepared by the random priming method. PCR products were analyzedby electrophoresis on 1.5% agarose gels, transferred to nylon membranes,and probed with the appropriate ³²P-labeled DNA insert.

A recombinant clone was identified in the above assays which, followingsequence comparison of its predicted amino acid sequence with sequencesof other proteins, was identified as a Leishmania braziliensis homologof the eukaryotic 83 kD heat shock protein (Lbhsp83). The sequence ofthe clone is provided in SEQ ID NO:5 and the deduced protein sequence isprovided in SEQ ID NO:6. On the basis of the homology, this clone,designated Lbhsp83a, appears to lack the first 47 residues of the fulllength 703 amino acid residues. Lbhsp83 has an overall homology of 94%(91% identity and 3% conservative substitution), 91% (84% identity and7% conservative substitution) and 77% (61% identity and 16% conservativesubstitution) with L. amazonensis hsp83, T. cruzi hsp83 and human hsp89,respectively. A second clone (designated Lbhsp83b), which contained the43 kD C-terminal portion of hsp83 (residues 331 to 703) was alsoisolated. FIG. 19 presents a comparison of the Lbhsp83 sequence with L.amazonensis hsp83(Lahsp83), T. cruzi hsp83 (Tchsp83) and human hsp89(Huhsp89).

The results of proliferation assays using Lbhsp83a are shown in Table 1.Cells from all mucosal leishmaniasis (ML) patients proliferated stronglyin response to Lbhsp83a, with stimulation indices (SIs) ranging from 19to 558 (as compared to 20 to 1,634 for parasite lysate). Proliferationof PBMC from cutaneous leishmaniasis (CL) patients was variable andexcept for levels in two patients (IV and VII), levels weresignificantly lower than those of ML patients. By comparison, theproliferative responses of individuals with self-healing CL to Lbhsp83awere similar to those of individuals with ML. However, the responses ofall six self-healing individuals to Lbhsp83 were consistently higherthan those to Lbhsp83b. This suggests that PBMC from self-healing CLpatients preferentially recognize one or more T-cell epitopes locatedwithin the amino portion of Lbhsp83.

TABLE 1 In vitro Proliferation of PMBC from L. braziliensis-infectedIndividuals in Response to Lbhsp83 Mean [³H]thymidine Group andincorporation [10³ cpm (SD)], SI with: Patient Lysate Lbhsp83a Lbhsp83bML I 41.3, (1.3), 294 32.5, (6.6), 221 46.7, (1.4), 318 II 44.2, (0.5),104 20, (3.7), 47 36.7, (0.76), 86 III 27.4, (1.5), 150 8.1, (1.7), 449.9, (0.32), 54 IV 52.7, (3.3), 138 54.1, (6.2), 142 32.0, (1.3), 84 V140.6, (7.6), 308 151.8, (57), 333 150.4, (7.9), 331 VI 15.8, (1.8), 2021.3, (4.4), 28 14.4, (1.3), 19 VII 300.1, (9.4), 1634 102.1, (7.6), 55841.7, (4.9), 228 CL I 0.26, (0.0), 1.5 0.57, (0.3), 3.3 0.43, (0.17),3.3 II 55.63, (8.6), 218 0.42, (0.0), 1.6 0.8, (0.14), 3.2 III 0.39,(0.5), 4.0 3.4, (0.5), 9 2.6, (0.9), 6.6 IV 19.14, (1.3), 87 7.17,(0.6), 32 5.9, (0.9), 27 V 0.32, (0.2), 3.0 1.47, (0.5), 14 0.3, (0.1),3.0 VI 0.77, (0.1), 4.7 1.44, (0.2), 9 1.3, (0.6), 8.0 VII 4.01, (1.0),2.0 60.3, (8.5), 15 66.7, (3.9), 16.6 Self-healing CL I 19.7, (4.4), 9461.3, (4.6), 293 5.0, (2.0), 24 II 0.6, (0.1), 6.5 7.0, (2.0), 79 1.2,(0.8), 13 III 59.6, (7.1), 519 49.4, (3.1), 429 21.4, (3.7), 186 IV 0.2,(0.1), 1.6 13.1, (1.7), 108 0.6, (0.1), 5 V 27.1, (2.0), 225 6.3, (2.6),52 3.0, (1.5), 25 VI 130.3, (14), 340 28.2, (2.9), 74 7.7, (3.8), 20Control (uninfected) I 0.19, (0.0), 1.4 0.18, (0.0), 1.3 0.40, (0.16),2.8 II 0.31, (0.1), 1.7 0.19, (0.0), 1.0 0.27, (0.0), 1.5 III 0.44,(0.2), 4.1 0.48, (0.1), 5.0 0.51, (0.2), 5.2 IV 0.4, (0.1), 3.2 0.52,(0.2), 5.1 0.50, (0.1), 5.0

A more detailed analysis of cytokine patterns of PBMC from ML patientswas performed by reverse transcriptase PCR. Cytokine mRNAs wereevaluated in cells prior to culturing (FIG. 9, lanes O) or followingculturing in the absence (lanes −) or presence of the indicated antigenfor 48 and 72 h. FIG. 4A shows the results for five of the six MLpatients whose PBMC were analyzed. In about half of the ML patients,noncultured (resting) PBMC had detectable levels of mRNA for IFN-γ,IL-2, and IL-4 but not IL-10. CL patient PBMC, however, had IL-10 mRNAin the resting state in addition to mRNAs for the other cytokines tested(FIG. 4B). Following in vitro culture without antigen, the levels ofmRNA for IFN-γ, IL-2, and IL-4 in resting cells from ML patientsdecreased to background levels while IL-10 mRNA levels increased. Incontrast, PBMC of most CL patients had stable or increased IL-10 mRNA,while the mRNAs for IL-2, IFN-γ, and IL-4 were reduced to barelydetectable levels in the absence of antigen stimulation.

In PBMC of three ML patients, stimulation with lysate resulted inincreased expression of mRNA for IFN-Y, IL-2, and IL-4 but not IL-10. Bycomparison, both Lbhsp83 polypeptides elicited the production of mRNAfor IFN-γ and IL-2 from all ML patient PBMC tested. In contrast,profiles of mRNA for IL-10 and IL-4 differed for the two hsp83polypeptides. Lbhsp83a stimulated the production of IL-10 but not IL-4mRNA (patients I, II, III, and IV), while Lbhsp83b stimulated theproduction of IL-4 but not IL-10 mRNA in all six patients.

All CL patients tested responded to both Lbhsp83 polypeptides as well asto the parasite lysate by upregulating the synthesis of mRNAs for IL-2and IFN-γ, and in two of four patients (I and IV), the level of IL-4mRNA also increased, indicating stimulation of both Th1 and Th2cytokines. Interestingly and as in the case of ML patient unculturedPBMC which did not have detectable levels of IL-10 mRNA, Lbhsp83a andnot Lbhsp83b stimulated PBMC from one CL patient (IV) to synthesizeIL-10 mRNA. However, in the other three patients (I, II, and III) withresting levels of IL-10 mRNA, both rLbhsp83 polypeptides as well as theparasite lysate downregulated the expression of IL-10 mRNA.

PBMC supernatants were also assayed for the presence of secreted IFN-γ,TNF-α, IL-4, and IL-10. Cells from all ML and self-healing CL patients(seven and six patients, respectively) and from four of seven CLpatients were analyzed for secreted IFN-γ following stimulation withboth rLbhsp83 polypeptides, parasite lysate and Lbhsp70, an L.braziliensis protein homologous to the eukaryotic 70 kD heat shockprotein (FIG. 10A). In general, rLbhsp83a stimulated patient PBMC tosecrete higher levels of IFN-γ than did rLbhsp83b (0.2 to 36 and 0.13 to28 ng/ml, respectively). The presence of secreted IFN-γ correlated wellwith the corresponding mRNA detected by PCR.

PBMC from four of five ML patients (I, II, V, and VII) had supernatantTNF-α levels (0.8 to 2.2 ng/ml) higher than those detected in culturesof PBMC from uninfected controls following stimulation with parasitelysate (FIG. 10B). Similarly, the same PBMC were stimulated by rLbhsp83to produce levels of TNF-α in supernatant ranging from 0.61 to 2.9ng/ml. Compared with those of uninfected controls, PBMC from three (I,V, and VI), five (I, II, IV, V, and VI), and two (II and V) of sixindividuals analyzed produced higher levels of TNF-α in response toparasite lysate, rLbhsp83a, and rLbhsp83b, respectively. The levels ofTNF-α produced by PBMC from CL patients in response to parasite lysatewere comparable to those produced by uninfected controls. However,rLbhsp83 stimulated TNF-α production in the PBMC of two of thesepatients. rLbhsp83a stimulated higher levels of TNF-α, production thandid rLbhsp83b. In the absence of antigen stimulation, only PBMC from MLpatients (five of six) produced detectable levels of supernatant TNF-α(60 to 190 pg/ml).

In agreement with the IL-10 mRNA, IL-10 was detected by ELISA in theantigen-stimulated PMBC culture supernatants from ML and CL patients.The levels (49 to 190 pg) were significantly higher (up to 10-fold)following stimulation with rLbhsp83a compared with those after parallelstimulation of the same cells with rLbhsp83b (FIG. 11). Parasite lysatealso stimulated PMBC from some of the patients to produce IL-10.Although rLbhsp83 stimulated PMBC from uninfected individuals to produceIL-10, with one exception, the levels were lower than those observedwith patient PMBC. IL-4 was not detected in any of the supernatantsanalyzed. Therefore, the level of any secreted IL-4 is below thedetection limit of the ELISA employed (50 pg/ml). Taken together, theresults demonstrate that a predominant Th1 -type cytokine profile isassociated with PMBC from L. braziliensis-infected individuals followingstimulation with rLbhsp83 polypeptides.

To determine the correlation between the observed T-cell responses andantibody production to Lbhsp83, we compared the antibody (immunoglobulinG) reactivities to Lbhsp83 in sera from the three patient groups (FIG.12). The ELISA reactivities of ML patient sera with rLbhsp83a werecomparable to those observed with parasite lysate, and in general, therewas a direct correlation between ML patient anti-Lbhsp83 antibody titerand T-cell proliferation. Of 23 serum samples from ML patients analyzed,22 were positive (˜96%) with absorbance values of 0.20 to >3.0. Elevenof the ML patient serum samples had optical density values that were >1.In general, CL patients had significantly lower anti-Lbhsp83 antibodytiters (=0.74; standard error of the mean [SEM]=0.1) compared to thoseof ML patients. Therefore, ML and CL patient anti-rhsp83 antibody titerscorrelated with their respective T-cell proliferative responses.Anti-rLbhsp83 antibody titers were significantly higher in patients withML (=1.5; SEM=0.2) than in self-healing CL patients (=0.35; SEM=0.056),although their T-cell proliferative responses were similar. In fact,anti-Lbhsp83 antibody titers in serum from self-healing CL patients werecomparable to those from uninfected controls (=0.24; SEM=0.028). Byusing 2 standard deviations greater than the mean absorbance value ofuninfected control (0.484) as a criterion for positive reactivity toLbhsp83, eight of nine of the self-healing patient serum samples testedwere negative.

Example 4 Preparation Of Clones Encoding LT-210

This Example illustrates the preparation of clones encoding portions ofthe Leishmania antigen Lt-210, and which has the sequence provided inSEQ ID NO:8.

An expression library was constructed from L. tropica(MHOM/SA/91/WR1063C) genomic DNA. The DNA was isolated by solubilizingL. tropica promastigotes in 10 mM Tris-HCl, pH 8.3, 50 mM EDTA, 1% SDSand treating with 100 μg/ml RNaseA and 100 μg/ml proteinase K. Thesample was then sequentially extracted with an equal volume of phenol,phenol: chloroform (1:1), and Chloroform. DNA was precipitated by adding0.1 volume of 3M sodium acetate (pH 5.2) and 2.5 volume 95% ethanol. Theprecipitate was resuspended in 10 μM Tris, 1 mM EDTA. DNA was sheared bypassage through a 30-gauge needle to a size range of 2-6 kilobase, andwas repaired by incubation with DNA poli in the presence of 100 μM eachDATP, dCTP, dGTP, and dTTP. EcoRI adapters were ligated to the DNAfragments. After removal of unligated adapters by passage over a G-25Sephadex™ column, the fragments were inserted in EcoRI cut Lambda ZapII(Stratagene, La Jolla, Calif.).

Approximately 43,000 pfu were plated and screened with sera isolatedfrom viscerotropic leishmaniasis (VTL) patients. Sera from VTL patientswere received from Drs. M. Grogl and A. Magill. The VTL patient groupincluded eight individuals from whom parasites were isolated andcultured, seven of which had confirmed infection with L. tropica. Fourother patients were culture negative, but were still considered to beinfected based on either PCR analysis or a positive monoclonal antibodysmear (Dr. Max Grogl, personal communication). Serum samples from the 11infected patients were pooled and anti-E. coli reactivity removed byaffinity chromatography (Sambrook et al., supra, p. 12.27-12.28). Lambdaphage expressing reactive proteins were detected after antibody bindingby protein A-horseradish peroxidase and ABTS substrate.

Three clones, Lt-1, Lt-2, and Lt-3, containing a portion of the Lt-210gene were identified and purified. The clones ranged in size from 1.4 to3.3 kb and encoded polypeptides of 75 kD, 70 kD, and 120 kD,respectively. These three clones contain partial sequences of the Lt-210gene. Lt-1 and Lt-2 are overlapping clones and were chosen for furtherstudy.

The DNA sequences of Lt-1 and Lt-2 were determined. Exonuclease IIIdigestion was used to create overlapping deletions of the clones(Heinikoff, Gene 28:351-359, 1984). Single strand template was preparedand the sequence determined with Applied Biosystems Automated Sequencermodel 373A or by Sanger dideoxy sequencing. The sequence on both strandsof the coding portion of Lt-1 clone was determined. The partial sequenceof one strand of Lt-2 clone was determined.

SEQ ID NO:7 presents the DNA sequence of Lt-1, and SEQ ID NO:8 providesthe predicted amino acid sequence of the open reading frame. The DNAsequence of the coding portion of the Lt-1 clone includes a repeatednucleotide sequence at the 5′ portion of the clone containing eightcopies of a 99 bp repeat, three copies of a 60 bp repeat unit, which ispart of the larger 99 bp repeat, and 800 bp of non-repeat sequence. Thededuced amino acid sequence of the 99 bp repeat contains limiteddegeneracies. The mass of the predicted recombinant protein is 67,060Daltons. A database search of PIR with the predicted amino acid sequenceof the open reading frame yielded no significant homology to previouslysubmitted sequences. Predicted secondary structure of the repeat portionof the clone is entirely α-helical.

Sequence analysis of Lt-2 revealed that the 3′ portion of the cloneconsisted of a mixture of 60 and 99 bp repeats that were identical,excepting occasional degeneracies, to the 60 and 99 bp repeats observedin Lt-1. Collectively, the sequencing data suggest that Lt-1 and Lt-2are different portions of the same gene, Lt-2 being upstream of Lt-1,with possibly a small overlap.

Hybridization analysis confirmed that rLt-2 and rLt-1 containoverlapping sequences. Genomic DNAs of various Leishmania species wererestricted with a variety of enzymes, separated by agarose gelelectrophoresis, and blotted on Nytran membrane filter (Schleicher &Schuell, Keene, N.H.). Inserts from rLt-1 and rLt-2 were labeled with³²P CTP by reverse transcriptase from random oligonucleotide primers andused as probes after separation from unincorporated nucleotides on aSephadex G-50 column. Hybridizations using the rLt-1 or the rLt-2 probeare performed in 0.2M NaH₂PO₄/3.6 M NaCl at 65° C., whereashybridization using the rLt-lr probe is performed in 0.2 M NaH₂PO₄/3.6 MNaCl/0.2 M EDTA at 60° C. overnight. Filters are washed in 0.075 MNaCl/0.0075 M sodium citrate pH 7.0 (0.15 M NaCl/0.0150 M sodium citratefor the Lt-lr probe), plus 0.5% SDS at the same temperature ashybridization.

Genomic DNA from a number of Leishmania species including L. tropicawere analyzed by Southern blots as described above using the Lt-1, Lt-2,and Lt-1r inserts separately as probes. Collectively, various digests ofL. tropica DNA indicate that this gene has a low copy number. A similar,overlapping pattern was observed using either the Lt-1 or Lt-2 insert asa probe, consistent with the premise that these two clones containsequences near or overlapping one another. In addition, sequenceshybridizing with these clones are present in other Leishmania species.

L. tropica isolates have limited heterogeneity. Southern analyses ofdigested genomic DNA from four L. tropica parasite strains isolated fromVTL patients and three L. tropica parasite strains isolated from CLcases (two human, one canine) were performed. The Lt-1r insert describedbelow was labeled and used as a probe. The seven different L. tropicaisolates yielded similar intensities and restriction patterns, with onlya single restriction fragment length polymorphism among the isolates.These data, along with Southern analyses with additional enzymes,indicate limited heterogeneity in this region among the L. tropicaisolates.

The recombinant proteins of Lt-1 and Lt-2 were expressed and purified.The nested deletion set of Lt-1 formed for sequencing included a clonereferred to as Lt-1r, which contains one and one-third repeats. Thispolypeptide was also expressed and purified. In vivo excision of thepBluescript SK⁻ phagemid from Lambda Zap II was performed according tothe manufacturer's protocol. Phagemid virus particles were used toinfect E. coli XL-1 Blue. Production of protein was induced by theaddition of IPTG. Protein was recovered by first lysing pellets ofinduced bacteria in buffer (LB, 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 10mM EDTA) using a combination of lysozyme (750 μg/mL) and sonication.rLt-1, rLt-2, and rLt-1r, were recovered from the inclusion bodies aftersolubilization in 8M urea (rLt-1 and rLt-2) or 4M urea (rLt-1r).Proteins rLt-1 and rLt-2 were enriched and separated by precipitationwith 25%-40% ammonium sulfate and rLt-1r was enriched by precipitationwith 10%-25% ammonium sulfate. The proteins were further purified bypreparative gel electrophoresis in 10% SDS-PAGE. Recombinant proteinswere eluted from the gels and dialyzed in phosphate-buffered saline(PBS). Concentration was measured by the Pierce (Rockford, Ill.) BCAassay, and purity assessed by Coomassie blue staining after SDS-PAGE.

Example 5 Preparation of LBEIF4A

This example illustrates the molecular cloning of a DNA sequenceencoding the L. braziliensis ribosomal antigen LbeIF4A.

A genomic expression library was constructed with sheared DNA from L.braziliensis (MHOM/BR/75/M2903) in bacteriophage λZAPII (Stratagene, LaJolla, Calif.). The expression library was screened with E.coli-preadsorbed patient sera from an L. braziliensis-infectedindividual with mucosal leishmaniasis. Plaques containing immunoreactiverecombinant antigens were purified, and the pBSK(−) phagemid excisedusing the manufacturer's protocols. Nested deletions were performed withExonuclease III to generate overlapping deletions for single strandedtemplate preparations and sequencing. Single stranded templates wereisolated following infection with VCSM13 helper phage as recommended bythe manufacturer (Stratagene, La Jolla, Calif.) and sequenced by thedideoxy chain terminator method or by the Taq dye terminator systemusing the Applied Biosystems Automated Sequencer Model 373A.

The immunoreactive recombinant antigens were then analyzed in patientT-cell assays for their ability to stimulate a proliferative andcytokine production, as described in Examples 7 and 8 below.

A recombinant clone was identified in the above assays which, followingsequence comparison of its predicted amino acid sequence with sequencesof other proteins, was identified as a Leishmania braziliensis homologof the eukaryotic initiation factor 4A (eIF4A). The isolated clone(pLeIF.1) lacked the first 48 amino acid residues (144 nucleotides) ofthe full length protein sequence. The pLeIF. 1 insert was subsequentlyused to isolate the full length genomic sequence.

SEQ ID NO:9 shows the entire nucleotide sequence of the full-lengthLbeIF4A polypeptide. The open reading frame (nucleotides 115 to 1323)encodes a 403 amino acid protein with a predicted molecular weight of45.3 kD. A comparison of the predicted protein sequence of LbeIF4A withthe homologous proteins from tobacco (TeIF4A), mouse (MeIF4A), and yeast(YeIF4A) shows extensive sequence homology, with the first 20-30 aminoacids being the most variable. The lengths (403, 413, 407, and 395 aminoacids), molecular weights (45.3, 46.8, 46.4, and 44.7 kDa), andisoelectric points (5.9, 5.4, 5.5, and 4.9) of LbeIF4A, TeIF4A, MeIF4Aand YeIF4A, respectively, are similar. LbeIF4A shows an overall homologyof 75.5% (57% identity, 18.5% conservative substitution) with TeIF4A,68.6% (50% identity, 18.6% conservative substitution) with MeIF4A and67.2% (47.6% identity, 19.6% conservative substitution) with YeIF4A.

Example 6 Preparation of Soluble Leishmania Antigens

This Example illustrates the preparation of soluble Leishmania antigensfrom an L. major culture supernatant. L. major promastigotes were grownto late log phase in complex medium with serum until they reached adensity of 2-3×10⁷ viable organisms per mL of medium. The organisms werethoroughly washed to remove medium components and resuspended at 2-3×10⁷viable organisms per mL of defined serum-free medium consisting of equalparts RPMI 1640 and medium 199, both from Gibco BRL, Gaithersburg, Md.After 8-12 hours, the supernatant was removed, concentrated 10 fold anddialyzed against phosphate-buffered saline for 24 hours. Proteinconcentration was then determined and the presence of at least eightdifferent antigens confirmed by SDS- PAGE. This mixture is referred toherein as “soluble Leishmania antigens.”

Example 7 Comparison of Intepleukin-4 and Interferon-γ ProductionStimulated by Leishmani Antigens

This Example illustrates the immunogenic properties of the antigensprepared according to Examples 1, 2, 5 and 6, as determined by theirability to stimulate IL-4 and IFN-γ in lymph node cultures from infectedmice and in human PBMC preparations. Lymph node cultures for use inthese studies were prepared from L. major-infected BALB/c mice 10 daysafter infection, as described in Example 2. PBMC were prepared usingperipheral blood obtained from individuals with cured L. donovaniinfections who were immunologically responsive to Leishmania. Diagnosisof the patients was made by clinical findings associated with at leastone of the following: isolation of parasite from lesions, a positiveskin test with Leishmania lysate or a positive serological test.Uninfected individuals were identified based on a lack of clinical signsor symptoms, a lack of history of exposure or travel to endemic areas,and the absence of a serological or cellular response to Leishmaniaantigens. Peripheral blood was collected and PBMC isolated by densitycentrifugation through Ficoll™ (Winthrop Laboratories, New York).

Culture supernatants were assayed for the levels of secreted IL-4 andIFN-γ. IFN-γ was quantitated by a double sandwich ELISA using mouseanti-human IFN-γ mAb (Chemicon, Temucula, Calif.) and polyclonal rabbitanti-human IFN-γ serum. Human rIFN-γ(Genentech Inc., San Francisco,Calif.) was used to generate a standard curve. IL-4 was quantitated insupernatants by a double sandwich ELISA using a mouse anti-human IL-4mAb (M1) and a polyclonal rabbit anti-human IL-4 sera (P3). Human IL-4(Immunex Corp., Seattle, Wash.) was used to generate a standard curveranging from 50 pg/ml to 1 ng/ml.

FIGS. 13A and 13B, illustrate the mean level of secreted IL-4 and IFN-γ,respectively, 72 hours after addition of 10 μg/mL of each of thefollowing antigens to a lymph node culture prepared as described above:soluble Leishmania antigen (i.e., an extract prepared from rupturedpromastigotes which contains membrane and internal antigens (SLA)),Ldp23, LbeIF4A (LeIF), Lbhsp83, M15 and LmeIF (the L. major homolog ofLbeIF4A). The levels of secreted IL-4 and IFN-γ in medium alone (i.e.,unstimulated) are also shown. While SLA elicits a predominantly Th2response from lymph node cells of Leishmania-infected mice, Ldp23,LbeIF4A, Lbhsp83 and M15 elicited relatively little IL-4 and largeamounts of IFN-γ, consistent with a Th1 response profile.

FIG. 14 shows the level of secreted IFN-γ in culture filtrate frominfected and uninfected human PBMC preparations 72 hours after additionof 10 μg/mL L. major lysate, M15 or L-Rack, an immunodominantleishmanial antigen in murine leishmaniasis. Similarly, FIG. 15illustrates the level of secreted IFN-γ in culture filtrate frominfected and uninfected human PBMC preparations 72 hours after additionof 10 μg/mL L. major lysate, soluble Leishmania antigens (prepared asdescribed in Example 6) or L-Rack. These results indicate that M15 andsoluble Leishmania antigens, but not L-Rack, are potent stimulators ofIFN-γ production in patient PBMC, but not in PBMC obtained fromuninfected individuals. Thus, M15 and soluble Leishmania antigens elicita dominant Th1 cytokine profile in both mice and humans infected withLeishmania.

Example 8 Comparison of Proliferation Stimulated by Leishmania Antigens

This Example illustrates the immunogenic properties of the antigensprepared according to Examples 1, 2, 5 and 6, as determined by theirability to stimulate proliferation in lymph node cultures from infectedmice and in human PBMC preparations.

For in vitro proliferation assays, 2-4×10⁵ cells/well were cultured incomplete medium (RPMI 1640 supplemented with gentamycin, 2-ME,L-glutamine, and 10% screened pooled A+ human serum; Trimar, Hollywood,Calif.) in 96-well flat bottom plates with or without 10 μg/ml of theindicated antigens or 5 μg/ml PHA (Sigma Immunochemicals, St. Louis,Mo.) for five days. The cells were then pulsed with 1 μCi of [³H]thymidine for the final 18 hours of culture.

FIG. 16 illustrates the proliferation observed after addition of 10μg/mL or 20 μg/mL of each of the following antigens to a lymph nodeculture prepared as described in Example 7: SLA, Ldp23, LbeIF4A,Lbhsp83, and M15. The level of proliferation without the addition ofantigen is also shown. Data are represented as mean cpm. These resultsdemonstrate that a variety of leishmanial antigens are capable ofstimulatory lymph node cell proliferation from Leishmania-infected mice.

FIGS. 17 and 18 illustrate the proliferation observed in human PBMCpreparations from Leishmania-immune and uninfected individuals followingthe addition of 10 μg/mL M15 and soluble Leishmania antigens,respectively. These values are compared to the proliferation observedfollowing the addition of culture medium, L. major lysate or L-Rack. Theresults show that M15 and soluble Leishmania antigens stimulateproliferation in Leishmania-immune PBMC, but not in PBMC obtained fromuninfected individuals, demonstrating that M15 and soluble antigens (butnot L-Rack) are recognized by PBMC from individuals immune to Leishmaniadue to a previous infection.

Example 9 Preparation of Lmsp1A and Lmsp9A

This Example illustrates the preparation of two soluble Leishmaniaantigens, Lmsp1a and Lmsp9a.

A. Purification of Lmsp1a and Lmsp9a from a Mixture of Soluble L. majorAntigens

A high titer rabbit sera was raised against L. major soluble antigens,prepared as described above in Example 6. Specifically, a New Zealandwhite rabbit was immunized subcutaneously at multiple sites with 180 μgof L. major soluble antigens in a suspension containing 100 μg muramyldipeptide and 50% incomplete Freund's adjuvant. Six weeks later therabbit was given a subcutaneous boost of 100 μg of the same solubleantigen preparation in incomplete Freund's adjuvant. This was followedby two intravenous boosts spaced two weeks apart, each with 100 μg ofthe soluble antigen preparation. Sera was collected from the rabbit 11days after the final boost.

Anti E. coli antibody reactivities were removed from the rabbit sera bypre-adsorbing on nitrocellulose filters containing lysed E. coli.Adsorbed sera were evaluated by Western blot analysis using 10 μgLeishmania promastigote lysate (lane 1) and 1 μg soluble L. majorantigen mixture (lane 2). As shown in FIG. 20, the rabbit sera was foundto be reactive with seven dominant antigens of the soluble L. majorantigen mixture with molecular weights ranging from 18 to >200 kDa. Afour times longer exposure of the same blot revealed three additionalimmunoreactive species with molecular weights less than 18 kDa. The samesera reacted with approximately 10 antigens of the promastigote lysate,but with a pattern significantly different from that observed with thesoluble L. major antigens (FIG. 20). This is suggestive of potentialpost-translational modification of the same antigen before(intracellular localization) and after secretion/shedding. Suchmodifications may include cleavage of a leader sequence and/or theaddition of carbohydrate molecules to the secreted/shed antigens.

The rabbit sera described above was subsequently used to screen an L.major cDNA expression library prepared from L. major promastigote RNAusing the unidirectional Lambda ZAP (uni-ZAP) kit (Stratagene) accordingto the manufacturer's protocol. A total of 70,000 pfu of the amplifiedcDNA library was screened with the rabbit sera at a 1:250 dilution.Nineteen positive clones were confirmed in the tertiary screening. Thephagemid were excised and DNA from each of the 19 clones was sequencedusing a Perkin Elmer/Applied Biosystems Division automated sequencerModel 373A. All 19 clones were found to represent two distinctsequences, referred to as Lmsp1a and Lmsp9a. The determined cDNAsequences for Lmsp1a and Lmsp9a are provided in SEQ ID NO: 19 and 21,respectively, with the corresponding amino acid sequences being providedin SEQ ID NO: 20 and 22, respectively.

B. Characterization of Lmsp1a and Lmsp9a

FIG. 21 shows the full-length cDNA (SEQ ID NO: 19) and predicted aminoacid sequence (SEQ ID NO: 20) for the antigen Lmsp1a. The EcoRI/XhoIinsert is 1019 bp long and contains the following features: a) the last17 nt of the spliced leader sequence characteristic of all trypanosomanuclearly encoded mRNA; b) 39 nt of 5′ untranslated sequence; c) an openreading frame of 453 nt long coding for a 151 deduced amino acidsequence with a predicted molecular mass of 16.641 kDa; and d) 471 nt of3′ untranslated sequence terminating with a poly A tail. The predictedamino acid sequence contains three potential phosphorylation sites atamino acid residues 3, 85 and 102. In addition, Lmsp1a contains an RGDsequence at residue 104, a sequence that may play a role in parasiteinvasion of the macrophage. RGD sequences have been shown to mediate thebinding of various adhesion proteins to their cell surface receptors.There is no obvious leader sequence (secretory signal) at the aminoterminal portion suggesting that the protein might be shed or excreted.Lmsp1a appears to be one of the most abundant antigens found in theculture supernatant of live promastigote, since 17 of the 19 clonescontain sequences of variable lengths identical to Lmsp1a.

Comparison of the amino acid sequence of Lmps1a with known sequencesusing the DNA STAR system (Version 87) revealed that Lmsp1a sharesbetween 65% to 70% homology with the eukaryotic nucleoside diphosphatekinase protein, also referred to in the mouse and human as a tumormetastasis inhibitor gene.

Southern blot analysis of genomic DNA from L. major (Friedlander strain)digested with a panel of restriction enzymes (lanes 1 to 7) and sixother Leishmania species of different geographic locations digested withPstI (lanes 8 to 13) using the full-length cDNA insert of Lmps1a,demonstrated that Lmsp1a is present in all the species characterizedwith a high degree of conservation (FIG. 22). This suggests evolutionarysignificance for the maintenance of Lmsp1a and the existence ofhomologous species among all the Leishmania species.

The remaining two cDNA clones isolated from the soluble L. major antigenmixture represent identical sequences (referred to as Lmsp9a; SEQ ID NO:21), suggesting that the two copies resulted from amplification of theprimary library. Sequencing of the Lmsp9a cDNA revealed that the clonedoes not contain the full length 5′ sequence since it is lacking boththe spliced leader and 5′ untranslated sequences. The 3′ end of the cDNAcontains a poly A stretch, as would be expected for a Leishmania mRNA.Of the predicted translated sequence (SEQ ID NO: 22), 34 of the 201amino acids (17%) represent cysteine residues. Comparison of thepredicted protein sequence with those of known proteins as describedabove, revealed some homology with other cysteine rich proteins such asthe major surface trophozoite antigen of Giardia lamblia and furinproteases.

Example 10 Preparation and Characterization of MAPS-1A

This Example illustrates the preparation and characterization of theLeishmania antigen MAPS-1A (SEQ ID NO: 24).

A pool of sera was obtained from 5 BALB/c mice that had been given aprimary immunization and two boosts with crude L. major promastigoteculture supernatant as described below in Example 12. These mice weresubsequently shown to be protected when challenged with a dose of liveL. major promastigotes generally found to be lethal. The mouse sera thusobtained were used to screen an L. major amastigote cDNA expressionlibrary prepared as described in Example 1. Several seroreactive cloneswere isolated and sequenced using a Perkin Elmer/Applied BiosystemsDivision automated sequencer Model 373A (Foster City, Calif.).

One of these clones, referred to herein as MAPS-1A, was found to befull-length. Comparison of the cDNA and deduced amino acid sequences forMAPS-1A (SEQ ID Nos: 23 and 24, respectively) with known sequences inthe gene bank using the DNA STAR system revealed no significanthomologies to known Leishmania sequences, although some sequencesimilarity was found to a group of proteins, known as thiol-specificantioxidants, found in other organisms.

Recombinant MAPS-1A protein having an amino-terminal HIS-Tag wasprepared using a high level E. coli expression system and recombinantprotein was purified by affinity chromatography as described inExample 1. Southern blot analysis of genomic DNA from L. major digestedwith a panel of restriction enzymes, seven other Leishmania speciesdigested with PstI, and two other infectious-disease pathogens (T. cruziand T. brucei), using the full length insert of MAPS-1A, demonstratedthat MAPS-1A is present in all eight Leishmania species tested (FIG.23). Northern blot analysis of L. major promastigote and amastigote RNAsindicated that MAPS-1A is constitutively expressed.

Using oligonucleotide primers (SEQ ID NOs:27 and 28) based on theMAPS-1A cDNA sequence provided in SEQ ID NO: 23, the corresponding genewas isolated from L. tropica by means of PCR (using 30 cycles of thefollowing temperature step sequence: 94° C., 1 minute; 50° C., 1 minute;72° C., 1 minute) The determined cDNA sequence for the L. tropicaMAPS-1A protein is provided in SEQ ID NO: 25, with the correspondingamino acid sequence being provided in SEQ ID NO: 26.

The ability of recombinant MAPS-1A to stimulate cell proliferation wasinvestigated as follows. PBMC from 3 L. braziliensis-infected patientshaving active mucosal leishmaniasis, from 4 patients post kala-azarinfection (previously infected with L. chagasi and/or L. donovani) andfrom 3 uninfected-individuals were prepared as described above inExample 7. The ability of MAPS-1A to stimulate proliferation of thesePBMC was determined as described in Example 8 above. As shown in FIG.24, significant levels of MAPS-1A specific PBMC proliferation were seenin 2 of the 7 Leishmania patients.

The ability of MAPS-1A to stimulate proliferation in mice lymph nodecultures was determined as described in Example 8. FIG. 25 shows theamount of proliferation stimulated by MAPS-1A (at 25 μg/ml, 5 μg/ml and1 μg/ml) as compared to that stimulated by the positive control ConA andby crude L. major promastigote supernatant proteins, 20 dayspost-infection with L. major. Cells isolated 20 days post-infection werehighly responsive to MAPS-1A, whereas cells isolated 10 dayspost-infection were unresponsive.

Example 11 Immunoreactivity of Soluble Leishmania Antigens with Serafrom Leishmania-infected Patients

The reactivity of MAPS-1A with sera from uninfected individuals, fromhuman leishmaniasis patients with cutaneous infection, from humanpatients with acute visceral leishmaniasis, and from L. major-infectedBALB/c mice was determined as follows.

Assays were performed in 96-well plates coated with 200 ng antigendiluted to 50 μL in carbonate coating buffer, pH 9.6. The wells werecoated overnight at 4° C. (or 2 hours at 37° C.). The plate contentswere then removed and the wells were blocked for 2 hours with 200 μL ofPBS/1% BSA. After the blocking step, the wells were washed five timeswith PBS/0.1% Tween 20™. 50 μL sera, diluted 1:100 in PBS/0.1% Tween20™/0.1% BSA, was then added to each well and incubated for 30 minutesat room temperature. The plates were then washed again five times withPBS/0.1% Tween 20™.

The enyzme conjugate (horseradish peroxidase—Protein A, Zymed, SanFrancisco, Calif.) was then diluted 1:10,000 in PBS/0.1% Tween 20™/0.1%BSA, and 50 μL of the diluted conjugate was added to each well andincubated for 30 minutes at room temperature. Following incubation, thewells were washed five times with PBS/0.1% Tween 20™. 100 μL oftetramethylbenzidine peroxidase (TMB) substrate (Kirkegaard and PerryLaboratories, Gaithersburg, Md.) was added, undiluted, and incubated forabout 15 minutes. The reaction was stopped with the addition of 100 μLof 1 N H₂SO₄ to each well, and the plates were read at 450 mn.

As shown in FIG. 26, approximately 50% of the samples from humanleishmaniasis patients showed reactivities with recombinant MAPS-1Asubstantially above background. FIG. 27 shows the reactivity of MAPS-1Awith increasing dilutions of sera from BALB/c mice previouslyadministered either (i) saline solution; (ii) the adjuvant B. pertussis;(iii) soluble Leishmania antigens plus B. pertussis; (iv) live L. majorpromastigotes; or (v) soluble Leishmania antigens plus B. pertussisfollowed by live L. major promastigotes (as described below in Example12). Considerably higher absorbances were seen with sera from miceinfected with live L. major promastigotes and with mice infected withlive L. major promastigotes following immunization with solubleLeishmania antigens plus B. pertussis, than with sera from the otherthree groups of mice, indicating that anti-MAPS-1A antibody titersincrease following Leishmania infection.

Example 12 Use of Leishmania Antigens for Vaccination Against LeishmaniaInfection

This example illustrates the effectiveness of Leishmania antigens inconferring protection against disease in the experimental murineleishmaniasis model system. For a discussion of the murine leishmaniasismodel system see, for example, Reiner et al. Annu. Rev. Immunol.,13:151-77, 1995.

The effectiveness of (i) crude soluble Leishmania antigens, (ii)MAPS-1A, and (iii) a mixture of Ldp23, LbeIF4A and M15, as vaccinesagainst Leishmania infection was determined as follows. BALB/c mice (5per group) were immunized intra-peritoneally three times at biweeklyintervals with either (i) 30 μg crude soluble Leishmania antigens, (ii)20 μg MAPS-1A or (iii) a mixture containing 10 μg each of LeIF, Ldp23and M15, together with 100 μg of the adjuvant C. parvum. Two controlgroups were immunized with either saline or C. parvum alone. Two weeksafter the last immunization, the mice were challenged with 2×10⁵late-log phase promastigotes of L. major. Infection was monitored weeklyby measurement of footpad swelling. The amount of footpad swelling seenin mice immunized with either crude soluble Leishmania antigens, amixture of Ldp23, LbeiF4A and M15 (FIG. 28), or MAPS-1A (FIG. 29) wassignificantly less than that seen in mice immunized with C. parvumalone. These results demonstrate that the Leishmania antigens of thepresent invention are effective in conferring protection againstLeishmania infection.

Example 13 Isolation of DNA Encoding for Soluble Antigens from an L.major Genomic DNA Library

This example illustrates the isolation of seven soluble Leishmaniaantigen genes from an L. major genomic DNA library.

An L. major genomic DNA expression library was prepared from L. majorpromastigotes using the unidirectional Lambda ZAP (uni-ZAP) kit(Stratagene) according to the manufacturer's protocol. This library wasscreened with a high titer rabbit sera raised against L. major solubleantigens, as described above in Example 9. Seven positive clones wereidentified. The phagemid were excised and DNA from each of the sevenclones was sequenced using a Perkin Elmer/Applied Biosystems Divisionautomated sequencer Model 373A. The DNA sequences for these antigens,referred to as LmgSP1, LmgSP3, LmgSP5, LmgSP8, LmgSP9, LmgSP13, LmgSP19,are provided in SEQ ID NO:29-35, respectively, with the correspondingamino acid sequences being provided in SEQ ID NO: 36-42, respectively.LmgSP13 was found to contain a 39 amino acid repeat sequence shown inSEQ ID NO:43.

Subsequent studies led to the isolation of an extended cDNA sequence forLmgSP13 which contains an ORF (cDNA sequence provided in SEQ ID NO: 114)encoding a 194 amino acid sequence (SEQ ID NO: 119). Comparison of thesesequences with those in the public databases revealed that LmgSP13encodes a portion of a clone recently identified in the L. major genomesequencing project (genomic DNA sequence provided in SEQ ID NO: 115).The full-length ORF encodes a 2310 amino acid polypeptide sequence(provided in SEQ ID NO: 120) containing unique amino- andcarboxy-terminal regions flanking 42 highly related 39 amino acidrepeats.

The 5 repeats of the original LmgSP 13 clone were subcloned into amodified pET vector both alone and as a fusion downstream of the M.tuberculosis antigen Ra12. The resulting recombinant protein wasexpressed, purified and used to generate a highly specific rabbitantiserum. Western blotting indicated that L. chagasi contains a LmgSp13homologue.

Subsequent studies resulted in the isolation of an extended sequence forLmgSP9. The extended DNA sequence is provided in SEQ ID NO: 54, with thecorresponding predicted amino acid sequence being provided in SEQ ID NO:55. The amino acid sequence was found to contain six 14 amino acidrepeat units (SEQ ID NO: 56), with each unit being further divided intotwo 7 amino acid units, provided in SEQ ID NO: 57 and 58. Comparison ofthe isolated sequences for LmgSP9 with sequences in the publicdatabases, revealed that LmgSP9 encodes for the carboy-terminal regionof a larger DNA sequence (SEQ ID NO: 116) that encodes a 708 amino acidpolypeptide (SEQ ID NO: 121) identified in the L. major genomesequencing project. LmgSP9 was found to share low homology with serineprotease, endo-protease furin and the major surface-labeled trophozoiteantigen of Giardia lamblia (25-30% identity). Surface localization ofLmgSP9 is consistent with motif predictions of an amino-terminal signalsequence, carboxy-terminal transmembrane domain and GPI anchor. Southernhybridization using the original LmgSP9 clone indicated that homologoussequences are present in all tested Leishmania species. Theamino-terminal 295 amino acids of LmgSP9 excluding the signal sequence(referred to as LmgSP9N-ht; cDNA sequence provided in SEQ ID NO: 117 andamino acid sequence provided in SEQ ID NO: 122) were subcloned into amodified pET vector and recombinant protein was expressed and purified.

Comparison of the DNA and amino acid sequences for the isolated antigensas described above, revealed no significant homologies to LmgSP1 andLmgSP3. LmgSP5 was found to be related to the known Promastigote surfaceantigen-2 (PSA2) family. LmgSP8 was found to bear some homology to asequence previously identified in E. coli(2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid synthase).LmgSP9 and LmgSP 19 were found to be homologous to a L. majorhydrophilic surface protein referred to as Gene B (Flinn, H. M. et al.Mol. Biochem. Parasit. 65:259-270, 1994), and to ubiquitin,respectively. To the best of the inventors' knowledge, none of theseantigens have been previously shown to elicit T or B cell responses.

In further studies, a 220 bp DNA fragment was amplified from LmgSP5 andused to screen a L. major genomic library in Lambda ZAP. Seventeenpositive clones were purified after secondary screening. To select for aclone that had a likelihood of having the 5′ sequence of the LmgSP5insert, a labeled oligonucleotide from the 5′ region was used to screenthe DNA from the secondary positive clones. DNA from three cloneshybridized to the 5′ oligonucleotide, with one clone hybridizingstronger than the other two. This clone (cDNA sequence provided in SEQID NO: 103) was found to contain an insert of 2421 bp which containedthe entire open reading frame for the novel PSA-2 gene. This ORF wasamplified and cloned in the expression vector pET-17b for expression ofrecombinant protein in E. coli. The cDNA sequence of the ORF is providedin SEQ ID NO: 102, with the corresponding amino acid sequence beingprovided in SEQ ID NO: 104.

The reactivity of recombinant LmgSP9 with sera from patients withvisceral leishmaniasis, (from both Sudan and Brazil) and from normaldonors was evaluated by ELISA as described above. The absorbance valueswere compared with those obtained using the known Leishmania antigen K39described above, with L. chagasi lysate being employed as a positivecontrol. Representative results of these assays are provided below inTable 2, wherein all the patients from Brazil and those from the Sudandesignated as “VL” were inflicted with visceral leishmaniasis. Theresults demonstrated that LmgSP9 specifically detects antibody in mostindividuals with visceral leishmaniasis, regardless of geographicallocation. In several cases, the absorbance values of the antibodyreactivity to LmgSP9 were comparable to that observed with K39. Inaddition, LmgSP9 detected several cases of leishmaniasis that were notdetected using K39. These results indicate that LmgSP9 can be used tocomplement the reactivity of K39.

TABLE 2 REACTIVITY OF LMGSP9 WITH SERA FROM LEISHMANIA PATIENTS PatientNo. L. chagasi lysate K39 LmgSP9 Sudanese samples: B19 1.067 0.306 0.554B25 1.884 3.435 0.974 B43 1.19  3.225 0.86  B47 2.405 2.892 0.375 B500.834 0.748 0.432 B58 0.921 0.235 0.92  B63 1.291 0.303 0.764 B70 0.3170.089 3.056 VL4 1.384 3.035 2.965 VL11 0.382 0.144 0.142 VL12 0.2770.068 0.098 VL13 0.284 0.12  0.194 Brazilian samples: 105 3.508 3.53 0.374 106 2.979 3.373 2.292 107 2.535 3.444 0.46  109 1.661 3.415 3.319111 3.595 3.537 0.781 112 2.052 3.469 0.63  113 3.352 3.429 0.963 1142.316 3.437 1.058 115 2.073 3.502 1.186 116 3.331 3.461 0.96  NormalDonors: 129 0.157 0.104 0.08  130 0.195 0.076 0.095 131 0.254 0.1340.086 132 0.102 0.035 0.043

In subsequent ELISA analyses performed as described above, LmgSP 13 wasdemonstrated to react as strongly with sera from patients infected withL. chagasi as with sera from L. major infected patients. This isconsistent with the Western blot studies discussed above wherein L.chagasi was found to contain an LmgSp13 homologue.

In order to obtain a higher specificity for the detection of antibodiesin sera from visceral leishmaniasis patients, a homologue of LmgSP9 wasisolated from L. chagasi, one of the causative agents of visceralleishmaniasis. A total of 80,000 pfu of an amplified L. chagasi genomiclibrary were screened with the entire coding region of LmgSP9 (amplifiedfrom L. major genomic DNA). Seven hybridizing clones were purified tohomogeneity. The determined DNA sequences for two of these clones,referred to as Lc Gene A and LcGene B, are provided in SEQ ID NO: 59 and60, respectively, with the corresponding predicted amino acid sequencesbeing provided in SEQ ID NO: 61 and 62, respectively. The open readingframe for Lc Gene A was found to show some homology to Gene A/C,previously isolated from L. major (McKlean et al., Mol. Bio. Parasitol.,85:221-231, 1997). The open reading frame for Lc Gene B showed somehomology to Gene B of L. major, discussed above, and was found tocontain eleven repeats of a 14 amino acid repeat unit (SEQ ID NO: 63),with each repeat being further divided into two 7 amino acid units,provided in SEQ ID NO: 64 and 65.

The diagnostic potentials of Lc Gene A and Lc Gene B were evaluated byELISA as described above using sera from visceral leishmaniasis patientsfrom Sudan and Brazil, and from uninfected controls. Absorbance valueswere compared to those obtained using LmgSP9. Much higher absorbancevalues were obtained with Lc Gene A and Lc Gene B than with LmgSP9, withLc Gene B appearing to be more effective that Lc Gene A in detectingantibodies in certain cases. These results indicate that Lc Gene B ishighly effective in the diagnosis of visceral leishmaniasis.

In order to assess the diagnostic potential of the repeats found withinLc Gene B, a series of 6 peptides were synthesized (SEQ ID NO: 66-71;referred to as Pep 1-6), differing in an R or H residue. An ELISA wascarried out using the full-length LcGene B protein and the six peptides.The absorbance values obtained with Pep 3 were higher than thoseobtained with the other 5 peptides, however they were not as high asthose obtained with the full length protein.

Example 14 Isolation and Characterization of DNA Encoding for SolubleAntigens from an L. chagasi Genomic DNA Library

This example illustrates the preparation of five soluble Leishmaniaantigen genes from an L. chagasi genomic DNA library.

An L. chagasi genomic DNA expression library was prepared from L.chagasi promastigotes using the unidirectional Lambda ZAP (uni-ZAP) kit(Stratagene) according to the manufacturer's protocol. This library wasscreened with a high titer rabbit sera raised against L. major solubleantigens, as described above in Example 9. Five positive clones wereidentified. The phagemid were excised and DNA from each of the Fiveclones was sequenced using a Perkin Elmer/Applied Biosystems Divisionautomated sequencer Model 373A. The DNA sequences for these antigens,referred to as LcgSP1, LcgSP3, LcgSP4, LcgSP8, and LcgSP10 are providedin SEQ ID NO:44-48, respectively, with the corresponding amino acidsequences being provided in SEQ ID NO:49-53, respectively.

Comparison of these sequences with known sequences in the gene bank asdescribed above, revealed no known homologies to LcgSP3, LcgSP4, LcgSP8and LcgSP10. LcgSP1 was found to be homologous to the known antigenHSP70.

Subsequent studies led to the isolation of a longer cDNA sequence forLcgSP3. This clone was found to contain an ORF (cDNA sequence providedin SEQ ID NO: 113) encoding an amino acid sequence of 539 residues (SEQID NO: 118). Comparison of the sequence for LcgSP3 with those in thepublic database, revealed it to be most closely related to thethermostable carboxypeptidase of Vibrio cholera (45% identity).Moreover, LcgSP3 was found to contain the active site residuescharacteristic of this class of carboxypeptidase. Southern hybridizationusing the LcgSP3 ORF indicated that homologous sequences are present inall tested Leishmania species. The LcgSP3 ORF was subcloned into amodified pET vector and recombinant protein was expressed, purified andused to generate a highly specific rabbit antiserum, using conventionaltechniques.

FIGS. 30A and B illustrate the proliferative response of murine lymphnodes to recombinant LcgSP8, LcgSP10 and LcgSP3. Lymph nodes were takenBALB/c mice 17 days after infection with L. major. Infection occurred byfootpad injection of 2×10⁶ parasites/footpad. The cells were stimulatedwith recombinant antigen and proliferation was measured at 72 hoursusing ³H-thymidine. FIG. 30A shows the CPM, a direct measurement ofmitotic activity in response to the antigens, and FIG. 30B shows thestimulation index, which measures the proliferative response relative tothe negative control.

Example 15 Isolation of DNA Encoding for L. major Antigens By CD4+ TCell Expression Cloning

This example illustrates the isolation of T cell antigens of L. majorusing a direct T cell screening approach.

Leishmania-specific CD4+ T cell lines were derived from the PBMC of anindividual who tested positive in a leishmania skin test but had noclinical history of disease. These T cell lines were used to screen a L.major amastigote cDNA expression library prepared as described inExample 1. Immunoreactive clones were isolated and sequenced asdescribed above. The determined cDNA sequences for the 8 isolated clonesreferred to as 1G6-34, 1E6-44, 4A5-63, 1B11-39, 2A10-37, 4G2-83, 4H6-41,8G3-100 are provided in SEQ ID NO: 72-79, respectively, with thecorresponding predicted amino acid sequences being provided in SEQ IDNO: 80-87, respectively. The cDNA sequences provided for 1E6-44,2A10-37, 4G2-83, 4H6-41 and 8G3-100 are believe to represent partialclones. All of these clones were shown to stimulate T cellproliferation.

Comparison of these sequences with those in the gene bank as describedabove revealed no known homologies to the antigen 4A5-63. 1G6-34 wasfound to have some homology to histone H2B previously identified in L.enrietti. Antigens 1E6-44, 1B11-39 and 8G3-100 showed some homology tosequences previously identified in other eukaryotes, in particularSaccharomyces cerevisae. 2A10-37 and 4H6-41 were found to be homologousto the two previously identified proteins alpha tubulin from L. donovaniand beta tubulin from L. major, respectively, and 4G2-83 was found to behomologous to elongation initiation factor 2 previously identified in T.cruzi.

Subsequent full-length cloning studies, using standard techniques, ledto the isolation of an extended cDNA sequence for 1E6-44, provided inSEQ ID NO: 105. The corresponding amino acid sequence is provided in SEQID NO: 106. An extended cDNA sequence for 2A10-37 is provided in SEQ IDNO: 107. This sequence was found to contain a complete open readingframe which encodes the amino acid sequence of SEQ ID NO: 108. Anextended cDNA sequence for 4G2-83 is provided in SEQ ID NO: 109. Thissequence contains a complete open reading frame which encodes the aminoacid sequence of SEQ ID NO: 110. An extended cDNA sequence for 8G3-100is provided in SEQ ID NO: 111, with the corresponding amino acidsequence being provided in SEQ ID NO: 112.

All eight of the antigens described above (1G6-34, 1E6-44, 4A5-63,1B11-39, 2A10-37, 4G2-83, 4H6-41, 8G3-100) were expressed in E. coli asrecombinant fusion proteins containing N-terminal histidine tags andwere purified to homogeneity using nickel affinity chromatography. All 8purified recombinant proteins elicited strong proliferative responsesfrom the CD4+ T cell lines employed in the library screening. T cellreactivity to 1G6-34, 4H6-41 and 8G3-100 was also observed in T cellsgenerated against both Leishmania promastigote culture filtrate andamastigote culture filtrate, indicating that these antigens areexpressed in both the promastigote and amastigote life stages at levelsthat are sufficient to evoke strong cellular immunes response.

The ability of the 8 antigens to stimulate proliferation and IFN-γproduction in PBMC from patients with active cutaneous leishmaniasis(CL) and from normal donors was examined as described above. In additionto the 8 antigens, leishmanial promastigote lysate (LPr) and purifiedprotein derivative from M. tuberculosis (PPD) were also tested. Thenumber of patients and/or donors responding to each antigen is shown inTable 3 below. All CL patients responded to at least one of the 8antigens. Most notably, the antigens 1G6-34 and 4H6-41 elicited cellproliferation in 6/7 and 7/7 CL patients, respectively, and IFN-γproduction in 6/7 and 5/7 CL patients, respectively. In addition 1G6-34was not recognized by PBMC from uninfected control donors.

TABLE 3 CELL PROLIFERATION AND IFN-γ PRODUCTION IN PBMC FROM PATIENTSWITH CUTANEOUS LEISHMANIASIS CL Patients Normal donors IFN-γ Cell IFN-γCell Antigen production Proliferation production Proliferation LPr 7/76/7 3/8 5/8 1G6-34 6/7 5/7 0/8 0/8 1E6-44 0/7 4/7 5/8 2/8 4A5-63 1/7 1/70/8 0/8 1B11-39 5/7 3/7 1/8 0/8 2A10-37 1/7 3/7 1/8 0/8 4H6-41 7/7 5/73/8 1/8 8G3-100 0/7 2/7 5/8 2/8 PPD 7/7 7/7 7/8 7/8

Example 16 Synthesis of Polypeptides

Polypeptides may be synthesized on a Perkin Elmer/Applied BiosystemsDivision 430A peptide synthesizer using FMOC chemistry with HPTU(O-Benzotriazole-N,N,N,′,N′-tetramethyluronium hexafluorophosphate)activation. A Gly-Cys-Gly sequence may be attached to the amino terminusof the peptide to provide a method of conjugation, binding to animmobilized surface, or labeling of the peptide. Cleavage of thepeptides from the solid support may be carried out using the followingcleavage mixture: trifluoroaceticacid:ethanedithiol:thioanisole:water:phenol (40:1:2:2:3). After cleavingfor 2 hours, the peptides may be precipitated in coldmethyl-t-butyl-ether. The peptide pellets may then be dissolved in watercontaining 0.1% trifluoroacetic acid (TFA) and lyophilized prior topurification by C18 reverse phase HPLC. A gradient of 0%-60%acetonitrile (containing 0.1% TFA) in water (containing 0.1% TFA) may beused to elute the peptides. Following lyophilization of the purefractions, the peptides may be characterized using electrospray or othertypes of mass spectrometry and by amino acid analysis.

Example 17 Use of Leishmania Antigens Plus Adjuvant for VaccinationAgainst Leishmania Infection

This example illustrates the effectiveness of recombinant Leishmaniaantigens, M15 and MAPS, plus an adjuvant, IL-12, in conferringprotection against disease in the experimental murine leishmaniasismodel system. For discussion of the murine leishmaniasis model systemsee, for example, Reiner et al., Annu. Rev. Immunol., 13:151 -77, 1995.The effectiveness of M15 and MAPS in combination with IL-12, as vaccineagainst Leishmania infection was determined as follows: BALB/c mice (5per group) were immunized subcutaneously in the left footpad, twice (3weeks apart) with the 10 μg of the individual antigens mixed with 1 μgof IL-12. As controls, three separate groups of mice were immunized withsoluble leishmania lysate antigens (SLA) plus IL-12, with IL-12 alone orwith PBS. Three weeks after the last immunization the mice were infectedin the right footpad with 2×10⁵ promastigote forms of L. major(stationary phase). Footpad swelling was then measured weekly. Resultsare expressed in FIG. 31 and clearly indicate that the mice immunizedwith either M15 or MAPS and IL-12 were greatly protected against theinfection; whereas mice immunized with IL-12 alone did not showprotection from infection. The protection induced by these antigens wasas efficient or better than that induced by SLA+IL-12, a regimen knownto induce good protection against leishmaniasis in this animal model(Afonso, L. C. C., T. M. Scharton, L. Q. Vieira, M. Wysocka, G.Trinchieri, and P. Scott. 1994. The adjuvant effect of interleukin-12 ina vaccine against Leishmania major. Science 263:235-237). The samepattern of protection described above, was obtained i.e., M15, MAPS, andSLA, induced protection against L. major infection when C. parvuminstead of IL-12 was used as adjuvant (Example 12). These resultsdemonstrate that both M15 and MAPS recombinant antigens induce excellentprotection against L. major infection in the BALB/c model of humanleishmaniasis. In addition, both antigens induced protection when testedin two different adjuvant formulations, (e.g., IL-12 and C. parvim.)This finding is of high significance because it demonstrates thatimmunity to leishmaniasis can be induced by the specific antigensdelivered in adjuvants that are suitable for human use.

Example 18 Use of Leishmania DNA for Vaccination Against LeishmaniaInfection

This example illustrates the effectiveness of Leishmania DNA inconferring protection against disease in the experimental murineleishmaniasis model system. For discussion of the murine leishmaniasismodel system see, for example, Reiner et al., Annu. Rev. Immunol.,13:151-77, 1995. The protection properties of the recombinant antigenswas tested by immunizing mice with naked DNA containing thecorresponding M15 and MAPS genes. The DNA construct used was thepcDNA3.1 vector (Invitrogen) containing a CMV promotor. BALB/c mice (5per group) were injected in the left footpad three times (3 weeks apart)with 100 g of the indicated naked DNA preparations. Mice were bledbefore and after the immunizations to monitor the development ofspecific immune response. The antibody response was evaluated by ELISA.Specific anti-M15 and anti-MAPS IgG2a antibodies were detected after thesecond immunization in the sera of the mice immunized with therespective naked DNA. The presence of specific antibodies indicates thatthe DNA immunization resulted in the production of specific proteinantigen. Three weeks after the last immunization, the mice were thenchallenged in the right footpad with 2×10⁵ promastigote forms of L.major (stationary phase). Footpad swelling was then measured weeklythereafter. Results are expressed in FIG. 32 and clearly indicated that,again, mice immunized with naked DNA containing either the M15 or MAPSgenes were greatly protected against the infection with L. major. Theseresults demonstrate that both M15 and MAPS genes induce excellentprotection against L. major infection in the BALB/c model of humanleishmaniasis.

Example 19 Preparation and Characterization of Leishmania FusionProteins

Fusion proteins comprising the Leishmania antigens MAPS-iA (SEQ ID NO:24), M15 (SEQ ID NO: 2), Lbhsp83 (SEQ ID NO: 6) and LbeIF4A (SEQ ID NO:10) were prepared as follows.

A fusion construct of MAPS-1A and M15 was prepared by first PCRamplifying the full-length coding sequence of MAPS-1A using the primersof SEQ ID NO: 88 and 89. The resulting products were digested with NdeIand BamHI follows by sub-cloning into the pET17b expression vector, alsodigested with NdeI and BamHI. The ligated products were transformed intoE. coli and transformants containing the correct insert were identifiedby restriction digest and verified by DNA sequencing. The MAPS-1A-pETplasmid was digested with BamHI and EcoRI. The latter cuts within thepoly-linker sequence of the pET vector which is located downstream ofthe BamHI site.

The primers of SEQ ID NO: 90 and 91 were employed to PCR amplify thefull-length coding sequence of M15 and the resulting product wasdigested with BamHI and EcoRI followed by sub-cloning into thepredigested MAPS1A-pET plasmid above. The ligated products were thentransformed into E. coli and transformants with the correct insert wereidentified by restriction digest and verified by DNA sequencing. TheMAPS1A-M15 pET construct was transformed into the bacterial host (BL21;pLysE). Expression of the protein resulted in a single recombinantmolecule with a predicted molecular weight of 85.7 kDa. The recombinantMAPS1A-M15 fusion protein also contained 33 amino acid residues ofrun-through vector as a result of the removal of the stop codon of M15and was subsequently digested with EcoRI. The DNA sequence of theMAPS1A-M15 construct is provided in SEQ ID NO: 101.

The primers of SEQ ID NO: 92 and 93 were used to PCR amplify the first226 amino acid residues of LbeIF4A. The resulting PCR product wasdigested with EcoRI and sub-cloned into the MAPS1A-M15-pET plasmid. Theligated products were then transformed into E. coli and transformantswith the correct insert and orientation were identified by restrictiondigest and verified by DNA sequencing. The expressed recombinant proteinwas purified by affinity chromatography over a Ni column. The DNA andamino acid sequences of the fusion protein MAPS1A-M15-LbeIF4A areprovided in SEQ ID NO: 94 and 95, respectively.

Additional fusion proteins were prepared using the methodology describedabove. The amino acid sequences for the fusion proteinsMAPS1A-M15-Lbhsp83 and MAPS1A-M15-Lbhsp83-LeIF4A are provided in SEQ IDNO: 96 and 97, respectively. The DNA sequence that encodes the aminoacid sequence of SEQ ID NO: 97 is provided in SEQ ID NO: 98. The DNAsequences of MAPS1A-M15-Lbhsp83 and MAPSlA-M15-Lbhsp83-LeIF4A vectorsemployed in DNA vaccines are provided in SEQ ID NO: 99 and 100,respectively.

Example 20 Use of Leishmania Fusion Proteins Plus Adjuvant forVaccination against Leishmania Infection

The ability of the Leishmania fusion proteins MAPS1A-M15 (referred to asthe diFusion) and MAPS1A-M15-LbeIF4A (referred to as the triFusion),plus adjuvant, to confer protection against disease in the experimentalmurine leishmaniasis model system was examined as follows.

The diFusion and triFusion were prepared as described above. In a firstseries of experiments, groups of BALB/c mice were immunized with eitherthe individual recombinant antigens, (MAPS1A, M15 or LbeIF4A), thediFusion or the triFusion, with IL-12 as an adjuvant, as described abovein Example 17. Control mice were immunized with IL-12 alone or saline.Before challenge, some mice (three per group) were sacrificed and theimmune responses to the fusion proteins and to the individual antigenswere investigated. Both T cell (cytokine production by spleen cells) andB cell responses (antibody response) were evaluated. The resultsindicated that immunization of mice with the fusion proteins did notinterfere with the immunogenicity of the individual antigens. Morespecifically, Th1 responses (namely induction of IFN-γ production andspecific IgG2a production) were observed to both MAPS1A and M15, whenmice were immunized with both the diFusion and triFusion recombinantproteins. In addition, immunization with the triFusion resulted in goodimmune response to LeIF.

To evaluate the protection conferred by these fusion proteins, theimmunized and control mice were infected in the right footpad with 2×10³amastigote forms of L. major and footpad swelling was measured weeklythereafter. The results, shown in FIG. 33, clearly indicated that bothfusion proteins induced protection comparable to MAPS1A and M15.

A second series of experiments was performed in which MPL-SE (RibiImmunoChem Research Inc. (Hamilton, Mont.) was employed as the adjuvant.BALB/c mice were immunized three times (three weeks interval) with 2 μgof the individual antigens (MAPS1A, M15 or LbeIF4A), diFusion ortriFusion proteins plus MPL-SE, and tested for immunogenicity of theantigens and for protection as described above. As with the experimentsperformed with IL-12 as adjuvant, the mice immunized with the individualantigens as well as with the fusion proteins showed both specific T andB cell responses to the immunizing antigens. Moreover, no antigencompetition between the individual antigens was observed when the fusionproteins were used as immunogens.

As with the protection studies in which IL-12 was used as adjuvant,protection was achieved with the individual antigens MAS1A and M15, aswell as with the two fusion proteins (FIG. 34). Slightly betterprotection was observed in the group of mice immunized with thetriFusion than in mice immunized with the diFusion.

Example 21 Formulation of Compositions Comprising Leishmania FusionProteins

A stable preparation of the tri-fusion of MAPS1A, M15 and LbeIF4Adescribed above was prepared as follows. The purified protein was putinto ammonium bicarbonate buffer (pH 8.0) by dialysis, and the followingwere added: 5% (w/v) mannitol, sucrose at 10:1 (w/w) excess sucrose toprotein and 0.1% (v/v) polysorbate 80. The protein was lyophilized todryness to yield a stable powder which can be readily resuspended asneeded.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for the purposeof illustration, various modifications may be made without deviatingfrom the spirit and scope of the invention.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 122 <210> SEQ ID NO 1 <211> LENGTH: 3134<212> TYPE: DNA <213> ORGANISM: Leishmania major <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(3134)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 1caagtgtcga aggacagtgt tcnccgtgtg agatcgccgg ctgtgcgtgt ga#aggcggtg     60ccatcggana aacaacaccg gtgganccgc aggaaaccat ctttctccgc ag#gtctcttt    120ttgttgtcga ttgagagtgc nccaaaccct gctggtgccc ttctcacata tc#atgttttt    180cgttgtgcgc tcgctttgcc tttcctctcc tttccctctc ttccgtggtg cc#gtgtatac    240ttctggcacc cgctacgtca cttcgctggt ttgaacagaa ccactgtgaa ca#cccacggg    300cgatcgcaca catacacatc cctcactcac acacacagct acatctatcc ta#cataaagc    360tgaaaaaaaa gtctacgaac aattttgttt ttacagtgcg ttgccgcaca tt#tctccgta    420atggacgcaa ctgagctgaa gaacaagggg aacgaagagt tctccgccgg cc#gctatgtg    480gaggcggtga actacttctc aaaggcgatc cagttggatg agcagaacag tg#tcctctac    540agcaaccgct ccgcctgttt tgcagccatg cagaaataca aggacgcgct gg#acgacgcc    600gacaagtgca tctcgatcaa gccgaattgg gccaagggct acgtgcgccg ag#gagcagct    660ctccatggca tgcgccgcta cgacgatgcc attgccgcgt atgaaaaggg gc#tcaaggtg    720gacccttcca acagcggctg cgcgcagggc gtgaaggacg tgcaggtagc ca#aggcccgc    780gaagcacgtg accccatcgc tcgcgtcttc accccggagg cgttccgcaa ga#tccaagag    840aatcccaagc tgtctctact tatgctgcag ccggactacg tgaagatggt ag#acaccgtc    900atccgcgacc cttcgcaggg ccggctgtac atggaagacc agcgctttgc cc#tgacgctc    960atgtacctga gcggaatgaa gattcccaac gatggtgatg gcgaggagga gg#aacgtccg   1020tctgcgaagg cggcagagac agcgaagcca aaagaggaga agcctctcac cg#acaacgag   1080aaggaggccc tggcgctcaa ggaggagggc aacaagctgt acctctcgaa ga#agtttgag   1140gaggcgctga ccaagtacca agaggcgcag gtgaaagacc ccaacaacac tt#tatacatt   1200ctgaacgtgt cggccgtgta cttcgagcag ggtgactacg acaagtgcat cg#ccgagtgc   1260gagcacggta tcgagcacgg tcgcgagaac cactgcgact acacaatcat tg#cgaagctc   1320atgacccgga acgccttgtg cctccagagg cagaggaagt acgaggctgc ta#tcgacctt   1380tacaagcgcg cccttgtcga gtggcgtaac cctgacaccc tcaagaagct ga#cggagtgc   1440gagaaggagc accaaaaggc ggtggaggaa gcctacatcg atcctgagat cg#cgaagcag   1500aagaaagacg aaggtaacca gtacttcaag gaggataagt tccccgaggc cg#tggcagcg   1560tacacggagg ccatcaagcg caaccctgcc gagcacacct cctacagcaa tc#gcgcggcc   1620gcgtacatca agcttggagc cttcaacgac gccctcaagg acgcggagaa gt#gcattgag   1680ctgaagcccg actttgttaa gggctacgcg cgcaagggtc atgcttactt tt#ggaccaag   1740cagtacaacc gcgcgctgca ggcgtacgat gagggcctca aggtggaccc ga#gcaatgcg   1800gactgcaagg atgggcggta tcgcacaatc atgaagattc aggagatggc at#ctggccaa   1860tccgcggatg gcgacgaggc ggcgcgccgg gccatggacg atcctgaaat cg#cggcaatc   1920atgcaagata gctacatgca actagtgttg aaggagatgc agaacgatcc ca#cgcgcatt   1980caggagtaca tgaaggactc cgggatctca tcgaagatca acaagctgat tt#cagctggc   2040atcattcgtt ttggtcagta gacttctacg ctgcctcatc ttttccgtgt ct#ttgcgtcg   2100gcgggtatcg taaagcacaa taaagcagcg attcacatgc acgagtaaag tg#ctgcgcct   2160ctcaaacacg acgtcgaggc tgtggtgcag atgcgcgtcc tgcatgaagg ta#gtgaagag   2220gaaagtaagg gatgttgttt gtgggccttc gtggctgcgc acacacctct ta#tctccttc   2280gcttggtacc ttctcccttt ttcgtcttca cccccctttc tcttctcacg ct#ctccctgg   2340cgcggtggtg caacgatttc gttttattta cgtctgtgta gctcctctat tc#aacggtgc   2400gatgacgcta acgaagctgg cctgtattcg gctaaggcga aggcaaaaga ct#aggagggg   2460ggggggaagg agacggcgtg accatcactg cgaagaaaca agccgaagaa aa#ggccccga   2520acgcctgcat ttccgcgcgc cctcgcccgc cttccttcct tccttcgctc tc#tctctctc   2580tctctctcgc tatcttctca acggagacat gaaaggcgtt tgttaggaaa ag#aggggggg   2640gggaagagtg ggacgacgcg ctgcgtcttt tgggcactgg tcacgtgcgt ca#ccctcttt   2700ttttatctct attggcactg tcttgtttct tttccctttc ctatcatacg cg#tctcgcaa   2760acgactccgc gctgagcagc catgtgctgc ggcgtggagg aagtacacag ac#atcacgga   2820tgcatatgtg cgcgtccgtg tacgcgcttg tatggggctt ctaacagcgc ct#gtgtgtgt   2880ttgtgtgtgt gtgtgtgtgt gtgtctgtgt atttcgagcg tctgtatgct at#tctattaa   2940gcaccgaaga agagacacac acgacagcga aggagatggt gtcggctttt cg#gctaatca   3000ctcccttcca tagcttctct gaaggaggct ctcttccaga ggaatagact gc#agatgggg   3060tccacgttta tctgaggagt caacggaaaa aaaaaaaaaa aaaaaaaaaa aa#aaaaaaaa   3120 aaaaaaaact cgag               #                  #                   #   3134 <210> SEQ ID NO 2 <211> LENGTH: 546<212> TYPE: PRT <213> ORGANISM: Leishmania major <400> SEQUENCE: 2Met Asp Ala Thr Glu Leu Lys Asn Lys Gly As #n Glu Glu Phe Ser Ala 1               5   #                10   #                15Gly Arg Tyr Val Glu Ala Val Asn Tyr Phe Se #r Lys Ala Ile Gln Leu            20       #            25       #            30Asp Glu Gln Asn Ser Val Leu Tyr Ser Asn Ar #g Ser Ala Cys Phe Ala        35           #        40           #        45Ala Met Gln Lys Tyr Lys Asp Ala Leu Asp As #p Ala Asp Lys Cys Ile    50               #    55               #    60Ser Ile Lys Pro Asn Trp Ala Lys Gly Tyr Va #l Arg Arg Gly Ala Ala65                   #70                   #75                   #80Leu His Gly Met Arg Arg Tyr Asp Asp Ala Il #e Ala Ala Tyr Glu Lys                85   #                90   #                95Gly Leu Lys Val Asp Pro Ser Asn Ser Gly Cy #s Ala Gln Gly Val Lys            100       #           105       #           110Asp Val Gln Val Ala Lys Ala Arg Glu Ala Ar #g Asp Pro Ile Ala Arg        115           #       120           #       125Val Phe Thr Pro Glu Ala Phe Arg Lys Ile Gl #n Glu Asn Pro Lys Leu    130               #   135               #   140Ser Leu Leu Met Leu Gln Pro Asp Tyr Val Ly #s Met Val Asp Thr Val145                 1 #50                 1 #55                 1 #60Ile Arg Asp Pro Ser Gln Gly Arg Leu Tyr Me #t Glu Asp Gln Arg Phe                165   #               170   #               175Ala Leu Thr Leu Met Tyr Leu Ser Gly Met Ly #s Ile Pro Asn Asp Gly            180       #           185       #           190Asp Gly Glu Glu Glu Glu Arg Pro Ser Ala Ly #s Ala Ala Glu Thr Ala        195           #       200           #       205Lys Pro Lys Glu Glu Lys Pro Leu Thr Asp As #n Glu Lys Glu Ala Leu    210               #   215               #   220Ala Leu Lys Glu Glu Gly Asn Lys Leu Tyr Le #u Ser Lys Lys Phe Glu225                 2 #30                 2 #35                 2 #40Glu Ala Leu Thr Lys Tyr Gln Glu Ala Gln Va #l Lys Asp Pro Asn Asn                245   #               250   #               255Thr Leu Tyr Ile Leu Asn Val Ser Ala Val Ty #r Phe Glu Gln Gly Asp            260       #           265       #           270Tyr Asp Lys Cys Ile Ala Glu Cys Glu His Gl #y Ile Glu His Gly Arg        275           #       280           #       285Glu Asn His Cys Asp Tyr Thr Ile Ile Ala Ly #s Leu Met Thr Arg Asn    290               #   295               #   300Ala Leu Cys Leu Gln Arg Gln Arg Lys Tyr Gl #u Ala Ala Ile Asp Leu305                 3 #10                 3 #15                 3 #20Tyr Lys Arg Ala Leu Val Glu Trp Arg Asn Pr #o Asp Thr Leu Lys Lys                325   #               330   #               335Leu Thr Glu Cys Glu Lys Glu His Gln Lys Al #a Val Glu Glu Ala Tyr            340       #           345       #           350Ile Asp Pro Glu Ile Ala Lys Gln Lys Lys As #p Glu Gly Asn Gln Tyr        355           #       360           #       365Phe Lys Glu Asp Lys Phe Pro Glu Ala Val Al #a Ala Tyr Thr Glu Ala    370               #   375               #   380Ile Lys Arg Asn Pro Ala Glu His Thr Ser Ty #r Ser Asn Arg Ala Ala385                 3 #90                 3 #95                 4 #00Ala Tyr Ile Lys Leu Gly Ala Phe Asn Asp Al #a Leu Lys Asp Ala Glu                405   #               410   #               415Lys Cys Ile Glu Leu Lys Pro Asp Phe Val Ly #s Gly Tyr Ala Arg Lys            420       #           425       #           430Gly His Ala Tyr Phe Trp Thr Lys Gln Tyr As #n Arg Ala Leu Gln Ala        435           #       440           #       445Tyr Asp Glu Gly Leu Lys Val Asp Pro Ser As #n Ala Asp Cys Lys Asp    450               #   455               #   460Gly Arg Tyr Arg Thr Ile Met Lys Ile Gln Gl #u Met Ala Ser Gly Gln465                 4 #70                 4 #75                 4 #80Ser Ala Asp Gly Asp Glu Ala Ala Arg Arg Al #a Met Asp Asp Pro Glu                485   #               490   #               495Ile Ala Ala Ile Met Gln Asp Ser Tyr Met Gl #n Leu Val Leu Lys Glu            500       #           505       #           510Met Gln Asn Asp Pro Thr Arg Ile Gln Glu Ty #r Met Lys Asp Ser Gly        515           #       520           #       525Ile Ser Ser Lys Ile Asn Lys Leu Ile Ser Al #a Gly Ile Ile Arg Phe    530               #   535               #   540 Gly Gln 545<210> SEQ ID NO 3 <211> LENGTH: 676 <212> TYPE: DNA<213> ORGANISM: Leishmania donovani <400> SEQUENCE: 3aattcggcac gaggcattgt gcataatggt caagtcccac tacatctgcg cg#ggccgcct     60ggtgcgcatc ctgcgtggcc cccgccagga ccgcgttggt gtgatcgtcg ac#attgtcga    120cgcgaaccgc gtgctggtgg agaacccgga ggacgcgaag atgtggcgcc ac#gtgcagaa    180cctgaagaac gtggagccgc tgaagtactg cgtgagcgtc agccgcaact gc#agcgcgaa    240ggcgctgaag gatgcgctgg cctcgtcgaa ggcgctggag aagtacgcga ag#acgcgcac    300tgctgcgcgc gtggaggcga agaaggcgtg cgccgcgtcg acggacttcg ag#cgctacca    360gctgcgcgtt gcgcgccgtt ctcgcgcgca ctgggcgcgc aaggtgttcg ac#gagaagga    420cgcgaagacg cccgtgtcgt ggcacaaggt tgcgctgaag aagatgcaga ag#aaggccgc    480aaagatggac tcgaccgagg gcgctaagag gcgcatgcag aaggcgatcg ct#gcccgcaa    540ggcgaaaaag taaggccata ccctcacttc gcttgtttcg tgatttttcg tg#ggagtcgg    600tggccctacc agcggtcttt cattggctta tttctatccg gtctgaaaga gg#tacaaaaa    660 aaaaaaaaaa aaaaaa              #                  #                   #   676 <210> SEQ ID NO 4 <211> LENGTH: 175<212> TYPE: PRT <213> ORGANISM: Leishmania donovani <400> SEQUENCE: 4Met Val Lys Ser His Tyr Ile Cys Ala Gly Ar #g Leu Val Arg Ile Leu 1               5   #                10   #                15Arg Gly Pro Arg Gln Asp Arg Val Gly Val Il #e Val Asp Ile Val Asp            20       #            25       #            30Ala Asn Arg Val Leu Val Glu Asn Pro Glu As #p Ala Lys Met Trp Arg        35           #        40           #        45His Val Gln Asn Leu Lys Asn Val Glu Pro Le #u Lys Tyr Cys Val Ser    50               #    55               #    60Val Ser Arg Asn Cys Ser Ala Lys Ala Leu Ly #s Asp Ala Leu Ala Ser65                   #70                   #75                   #80Ser Lys Ala Leu Glu Lys Tyr Ala Lys Thr Ar #g Thr Ala Ala Arg Val                85   #                90   #                95Glu Ala Lys Lys Ala Cys Ala Ala Ser Thr As #p Phe Glu Arg Tyr Gln            100       #           105       #           110Leu Arg Val Ala Arg Arg Ser Arg Ala His Tr #p Ala Arg Lys Val Phe        115           #       120           #       125Asp Glu Lys Asp Ala Lys Thr Pro Val Ser Tr #p His Lys Val Ala Leu    130               #   135               #   140Lys Lys Met Gln Lys Lys Ala Ala Lys Met As #p Ser Thr Glu Gly Ala145                 1 #50                 1 #55                 1 #60Lys Arg Arg Met Gln Lys Ala Ile Ala Ala Ar #g Lys Ala Lys Lys                165   #               170   #               175<210> SEQ ID NO 5 <211> LENGTH: 2040 <212> TYPE: DNA<213> ORGANISM: Leishmania braziliensis <400> SEQUENCE: 5cgcggtggcg gccgctctag aactagtgga tcccccgggc tgcaggaatt cg#gcacgaga     60gagcctgacg gacccggcgg tgctgggcga ggagactcac ctgcgcgtcc gc#gtggtgcc    120ggacaaggcg aacaagacgc tgacggtgga ggataacggc atcggcatga cc#aaggcgga    180cctcgtgaac aatctgggca cgatcgcgcg ctccggcacg aaggctttca tg#gaggcact    240ggaggccggc ggcgacatga gcatgatcgg ccagttcggt gtcggcttct ac#tccgcgta    300ccttgtggcg gaccgcgtga cggtggtgtc gaagaacaac tcggacgagg cg#tactggga    360atcgtctgcg gggggcacgt tcaccatcac gagcgtgcag gagtcggaca tg#aagcgcgg    420cacgagtaca acgctgcacc taaaggagga ccagcaggag tacctggagg ag#cgccgggt    480gaaggagctg atcaagaagc actccgagtt catcggctac gacatcgagc tg#atggtgga    540gaagacggcg gagaaggagg tgacggacga ggacgaggag gaggacgagt cg#aagaagaa    600gtcctgcggg gacgagggcg agccgaaggt ggaggaggtg acggagggcg gc#gaggacaa    660gaagaagaag acgaagaagg tgaaggaggt gaagaagacg tacgaggtca ag#aacaagca    720caagccgctc tggacgcgcg acacgaagga cgtgacgaag gaggagtacg cg#gccttcta    780caaggccatc tccaacgact gggaggacac ggcggcgacg aagcacttct cg#gtggaggg    840ccagctggag ttccgcgcga tcgcgttcgt gccgaagcgc gcgccgttcg ac#atgttcga    900gccgaacaag aagcgcaaca acatcaagct gtacgtgcgc cgcgtgttca tc#atggacaa    960ctgcgaggac ctgtgcccgg actggctcgg cttcgtgaag ggcgtcgtgg ac#agcgagga   1020cctgccgctg aacatctcgc gcgagaacct gcagcagaac aagatcctga ag#gtgatccg   1080caagaacatc gtgaagaagt gcctggagct gttcgaagag atagcggaga ac#aaggagga   1140ctacaagcag ttctacgagc agttcggcaa gaacatcaag ctgggcatcc ac#gaggacac   1200ggcgaaccgc aagaagctga tggagttgct gcgcttctac agcaccgagt cg#ggggagga   1260gatgacgaca ctgaaggact acgtgacgcg catgaagccg gagcagaagt cg#atctacta   1320catcactggc gacagcaaga agaagctgga gtcgtcgccg ttcatcgaga ag#gcgagacg   1380ctgcgggctc gaggtgctgt tcatgacgga gccgatcgac gagtacgtga tg#cagcaggt   1440gaaggacttc gaggacaaga agttcgcgtg cctgacgaag gaaggcgtgc ac#ttcgagga   1500gtccgaggag gagaagaagc agcgcgagga gaagaaggcg gcgtgcgaga ag#ctgtgcaa   1560gacgatgaag gaggtgctgg gcgacaaggt ggagaaggtg accgtgtcgg ag#cgcctgtt   1620gacgtcgccg tgcatcctgg tgacgtcgga gtttgggtgg tcggcgcaca tg#gaacagat   1680catgcgcaac caggcgctgc gcgactccag catggcgcag tacatggtgt cc#aagaagac   1740gatggaggtg aaccccgacc accccatcat caaggagctg cgccgccgcg tg#gaggcgga   1800cgagaacgac aaggccgtga aggacctcgt cttcctgctc ttcgacacgt cg#ctgctcac   1860gtccggcttc cagctggatg accccaccgg ctacgccgag cgcatcaacc gc#atgatcaa   1920gctcggcctg tcgctcgacg aggaggagga ggaggtcgcc gaggcgccgc cg#gccgaggc   1980agcccccgcg gaggtcaccg ccggcacctc cagcatggag caggtggact ga#gccggtaa   2040 <210> SEQ ID NO 6 <211> LENGTH: 656 <212> TYPE: PRT<213> ORGANISM: Leshmania brailiensis <400> SEQUENCE: 6Ser Leu Thr Asp Pro Ala Val Leu Gly Glu Gl #u Thr His Leu Arg Val  1               5  #                 10  #                 15Arg Val Val Pro Asp Lys Ala Asn Lys Thr Le #u Thr Val Glu Asp Asn             20      #             25      #             30Gly Ile Gly Met Thr Lys Ala Asp Leu Val As #n Asn Leu Gly Thr Ile         35          #         40          #         45Ala Arg Ser Gly Thr Lys Ala Phe Met Glu Al #a Leu Glu Ala Gly Gly     50              #     55              #     60Asp Met Ser Met Ile Gly Gln Phe Gly Val Gl #y Phe Tyr Ser Ala Tyr 65                  # 70                  # 75                  # 80Leu Val Ala Asp Arg Val Thr Val Val Ser Ly #s Asn Asn Ser Asp Glu                 85  #                 90  #                 95Ala Tyr Trp Glu Ser Ser Ala Gly Gly Thr Ph #e Thr Ile Thr Ser Val            100       #           105       #           110Gln Glu Ser Asp Met Lys Arg Gly Thr Ser Th #r Thr Leu His Leu Lys        115           #       120           #       125Glu Asp Gln Gln Glu Tyr Leu Glu Glu Arg Ar #g Val Lys Glu Leu Ile    130               #   135               #   140Lys Lys His Ser Glu Phe Ile Gly Tyr Asp Il #e Glu Leu Met Val Glu145                 1 #50                 1 #55                 1 #60Lys Thr Ala Glu Lys Glu Val Thr Asp Glu As #p Glu Glu Glu Asp Glu                165   #               170   #               175Ser Lys Lys Lys Ser Cys Gly Asp Glu Gly Gl #u Pro Lys Val Glu Glu            180       #           185       #           190Val Thr Glu Gly Gly Glu Asp Lys Lys Lys Ly #s Thr Lys Lys Val Lys        195           #       200           #       205Glu Val Lys Lys Thr Tyr Glu Val Lys Asn Ly #s His Lys Pro Leu Trp    210               #   215               #   220Thr Arg Asp Thr Lys Asp Val Thr Lys Glu Gl #u Tyr Ala Ala Phe Tyr225                 2 #30                 2 #35                 2 #40Lys Ala Ile Ser Asn Asp Trp Glu Asp Thr Al #a Ala Thr Lys His Phe                245   #               250   #               255Ser Val Glu Gly Gln Leu Glu Phe Arg Ala Il #e Ala Phe Val Pro Lys            260       #           265       #           270Arg Ala Pro Phe Asp Met Phe Glu Pro Asn Ly #s Lys Arg Asn Asn Ile        275           #       280           #       285Lys Leu Tyr Val Arg Arg Val Phe Ile Met As #p Asn Cys Glu Asp Leu    290               #   295               #   300Cys Pro Asp Trp Leu Gly Phe Val Lys Gly Va #l Val Asp Ser Glu Asp305                 3 #10                 3 #15                 3 #20Leu Pro Leu Asn Ile Ser Arg Glu Asn Leu Gl #n Gln Asn Lys Ile Leu                325   #               330   #               335Lys Val Ile Arg Lys Asn Ile Val Lys Lys Cy #s Leu Glu Leu Phe Glu            340       #           345       #           350Glu Ile Ala Glu Asn Lys Glu Asp Tyr Lys Gl #n Phe Tyr Glu Gln Phe        355           #       360           #       365Gly Lys Asn Ile Lys Leu Gly Ile His Glu As #p Thr Ala Asn Arg Lys    370               #   375               #   380Lys Leu Met Glu Leu Leu Arg Phe Tyr Ser Th #r Glu Ser Gly Glu Glu385                 3 #90                 3 #95                 4 #00Met Thr Thr Leu Lys Asp Tyr Val Thr Arg Me #t Lys Pro Glu Gln Lys                405   #               410   #               415Ser Ile Tyr Tyr Ile Thr Gly Asp Ser Lys Ly #s Lys Leu Glu Ser Ser            420       #           425       #           430Pro Phe Ile Glu Lys Ala Arg Arg Cys Gly Le #u Glu Val Leu Phe Met        435           #       440           #       445Thr Glu Pro Ile Asp Glu Tyr Val Met Gln Gl #n Val Lys Asp Phe Glu    450               #   455               #   460Asp Lys Lys Phe Ala Cys Leu Thr Lys Glu Gl #y Val His Phe Glu Glu465                 4 #70                 4 #75                 4 #80Ser Glu Glu Glu Lys Lys Gln Arg Glu Glu Ly #s Lys Ala Ala Cys Glu                485   #               490   #               495Lys Leu Cys Lys Thr Met Lys Glu Val Leu Gl #y Asp Lys Val Glu Lys            500       #           505       #           510Val Thr Val Ser Glu Arg Leu Leu Thr Ser Pr #o Cys Ile Leu Val Thr        515           #       520           #       525Ser Glu Phe Gly Trp Ser Ala His Met Glu Gl #n Ile Met Arg Asn Gln    530               #   535               #   540Ala Leu Arg Asp Ser Ser Met Ala Gln Tyr Me #t Val Ser Lys Lys Thr545                 5 #50                 5 #55                 5 #60Met Glu Val Asn Pro Asp His Pro Ile Ile Ly #s Glu Leu Arg Arg Arg                565   #               570   #               575Val Glu Ala Asp Glu Asn Asp Lys Ala Val Ly #s Asp Leu Val Phe Leu            580       #           585       #           590Leu Phe Asp Thr Ser Leu Leu Thr Ser Gly Ph #e Gln Leu Asp Asp Pro        595           #       600           #       605Thr Gly Tyr Ala Glu Arg Ile Asn Arg Met Il #e Lys Leu Gly Leu Ser    610               #   615               #   620Leu Asp Glu Glu Glu Glu Glu Val Ala Glu Al #a Pro Pro Ala Glu Ala625                 6 #30                 6 #35                 6 #40Ala Pro Ala Glu Val Thr Ala Gly Thr Ser Se #r Met Glu Gln Val Asp                645   #               650   #               655<210> SEQ ID NO 7 <211> LENGTH: 1771 <212> TYPE: DNA<213> ORGANISM: Leishmania tropica <400> SEQUENCE: 7caggcccgcg tccaggccct cgaggaggca gcgcgtctcc gcgcggagct gg#aggcggcc     60gaggaggcgg cccgcctgga tgtcatgcat gcggccgagc aggcccgtgt cc#aggccctc    120gaggaggcag cgcgtctccg cgcggagctg gaggaggccg aggaggcggc cc#gcctggat    180gtcatgcatg cggccgagca ggcccgcgtc caggccctcg aggaggcagc gc#gtctccgc    240gcggagctgg aggctgccga ggaggcggcg cgcctggagg ccatgcacga gg#ccgagcag    300gcccgctccc aggccctcga ggaggcagcg cgtctccgcg cggagctgga gg#aagccgag    360gaggcggccc gcctggatgt catgcatgcg gccgagcagg cccgcgtcca gg#ccctcgag    420gaggcagcgc gtctccgcgc ggagctggag gaggccgagg aggcggcccg cc#tggaggcc    480atgcacgagg ccgagcaggc ccgctcccag gccctcgagg aggcagcgcg tc#tccgcgcg    540gagctggagg cggccgagga ggcggcccgc ctggatgtca tgcacgaggc cg#agcaggcc    600cgtgtccagg ccctcgagga ggcggcgcgc ctggatgtca tgcacgaggc cg#agcaggcc    660cgcgtccagg ccctcgagga ggcagcgcgt ctccgcgcgg agctggaggc gg#ccgaggag    720gcggcccgcc tggatgtcat gcacgaggcc gagcaggccc gcgtccaggc cc#tcgaggag    780gcagcgcgtc tccgcgcgga gctggaggcg gccgaggagg cggcccgcct gg#atgtcatg    840cacgagggcg agcaggcccg tgtccaggcc ctcgaggagg cggcccgcct gg#aggccatg    900cacgaggccg agcaggcccg ctcccaggcc ctcgaggagg cagcgcgtct ct#gcgcggag    960ctggaggctg aggaggagga aaaagatgag cggccggcga cgtcgagcta ca#gcgaggag   1020tgcaaagggc gactgctatc gagggcgcgg ccggatccgc ggaggccgct gc#cgcggccg   1080ttcattggga tgtcactgtt ggaggatgtg gagaagagta ttctcattgt gg#acgggctc   1140tacagggatg ggccggcgta ccagacgggc atccgcctcg gggatgtcct ct#tgcgtatc   1200gcgggggttt acgtggattc aatagcgaag gcgaggcagg tggtcgatgc gc#gttgccgc   1260tgcggctgcg tcgttcccgt gacgctggcg acgaagatga accagcagta ca#gcgtggct   1320ctgtatatca tgacggtgga tccgcagcac aacgacaagc cctttttttt tg#atgtgcac   1380atccaccacc gcatcgagag ctcgcacatg gggaagaagg cgcagtggat gg#aagttctt   1440gagagcccat ccgtatcttc ggctgccacc acccctctcg tgccgctctt gc#gtgagccg   1500acgccgcgta ggggctcaga gctgcagtca agtgctcgtt ccgccttcgt tg#ccacgtct   1560tacttctcga gcgcgcgcag gtcggtcagc tcagaaagtg agcgaccgcg cg#ggtcctct   1620agcgtggcta tggcggagga ggcgatcgcg ctggcgccgc aagggtatac cc#cacccaac   1680caagtgcgcg gccgtagttg acgtctctgt gtgagtgtgt gtcgctccgt ct#ccttcctt   1740 tttcgtcatg tgttttattc atttcttttt c        #                   #        1771 <210> SEQ ID NO 8 <211> LENGTH: 566<212> TYPE: PRT <213> ORGANISM: Leishmania tropica <400> SEQUENCE: 8Gln Ala Arg Val Gln Ala Leu Glu Glu Ala Al #a Arg Leu Arg Ala Glu 1               5   #                10   #                15Leu Glu Ala Ala Glu Glu Ala Ala Arg Leu As #p Val Met His Ala Ala            20       #            25       #            30Glu Gln Ala Arg Val Gln Ala Leu Glu Glu Al #a Ala Arg Leu Arg Ala        35           #        40           #        45Glu Leu Glu Glu Ala Glu Glu Ala Ala Arg Le #u Asp Val Met His Ala    50               #    55               #    60Ala Glu Gln Ala Arg Val Gln Ala Leu Glu Gl #u Ala Ala Arg Leu Arg65                   #70                   #75                   #80Ala Glu Leu Glu Ala Ala Glu Glu Ala Ala Ar #g Leu Glu Ala Met His                85   #                90   #                95Glu Ala Glu Gln Ala Arg Ser Gln Ala Leu Gl #u Glu Ala Ala Arg Leu            100       #           105       #           110Arg Ala Glu Leu Glu Glu Ala Glu Glu Ala Al #a Arg Leu Asp Val Met        115           #       120           #       125His Ala Ala Glu Gln Ala Arg Val Gln Ala Le #u Glu Glu Ala Ala Arg    130               #   135               #   140Leu Arg Ala Glu Leu Glu Glu Ala Glu Glu Al #a Ala Arg Leu Glu Ala145                 1 #50                 1 #55                 1 #60Met His Glu Ala Glu Gln Ala Arg Ser Gln Al #a Leu Glu Glu Ala Ala                165   #               170   #               175Arg Leu Arg Ala Glu Leu Glu Ala Ala Glu Gl #u Ala Ala Arg Leu Asp            180       #           185       #           190Val Met His Glu Ala Glu Gln Ala Arg Val Gl #n Ala Leu Glu Glu Ala        195           #       200           #       205Ala Arg Leu Asp Val Met His Glu Ala Glu Gl #n Ala Arg Val Gln Ala    210               #   215               #   220Leu Glu Glu Ala Ala Arg Leu Arg Ala Glu Le #u Glu Ala Ala Glu Glu225                 2 #30                 2 #35                 2 #40Ala Ala Arg Leu Asp Val Met His Glu Ala Gl #u Gln Ala Arg Val Gln                245   #               250   #               255Ala Leu Glu Glu Ala Ala Arg Leu Arg Ala Gl #u Leu Glu Ala Ala Glu            260       #           265       #           270Glu Ala Ala Arg Leu Asp Val Met His Glu Gl #y Glu Gln Ala Arg Val        275           #       280           #       285Gln Ala Leu Glu Glu Ala Ala Arg Leu Glu Al #a Met His Glu Ala Glu    290               #   295               #   300Gln Ala Arg Ser Gln Ala Leu Glu Glu Ala Al #a Arg Leu Cys Ala Glu305                 3 #10                 3 #15                 3 #20Leu Glu Ala Glu Glu Glu Glu Lys Asp Glu Ar #g Pro Ala Thr Ser Ser                325   #               330   #               335Tyr Ser Glu Glu Cys Lys Gly Arg Leu Leu Se #r Arg Ala Arg Pro Asp            340       #           345       #           350Pro Arg Arg Pro Leu Pro Arg Pro Phe Ile Gl #y Met Ser Leu Leu Glu        355           #       360           #       365Asp Val Glu Lys Ser Ile Leu Ile Val Asp Gl #y Leu Tyr Arg Asp Gly    370               #   375               #   380Pro Ala Tyr Gln Thr Gly Ile Arg Leu Gly As #p Val Leu Leu Arg Ile385                 3 #90                 3 #95                 4 #00Ala Gly Val Tyr Val Asp Ser Ile Ala Lys Al #a Arg Gln Val Val Asp                405   #               410   #               415Ala Arg Cys Arg Cys Gly Cys Val Val Pro Va #l Thr Leu Ala Thr Lys            420       #           425       #           430Met Asn Gln Gln Tyr Ser Val Ala Leu Tyr Il #e Met Thr Val Asp Pro        435           #       440           #       445Gln His Asn Asp Lys Pro Phe Phe Phe Asp Va #l His Ile His His Arg    450               #   455               #   460Ile Glu Ser Ser His Met Gly Lys Lys Ala Gl #n Trp Met Glu Val Leu465                 4 #70                 4 #75                 4 #80Glu Ser Pro Ser Val Ser Ser Ala Ala Thr Th #r Pro Leu Val Pro Leu                485   #               490   #               495Leu Arg Glu Pro Thr Pro Arg Arg Gly Ser Gl #u Leu Gln Ser Ser Ala            500       #           505       #           510Arg Ser Ala Phe Val Ala Thr Ser Tyr Phe Se #r Ser Ala Arg Arg Ser        515           #       520           #       525Val Ser Ser Glu Ser Glu Arg Pro Arg Gly Se #r Ser Ser Val Ala Met    530               #   535               #   540Ala Glu Glu Ala Ile Ala Leu Ala Pro Gln Gl #y Tyr Thr Pro Pro Asn545                 5 #50                 5 #55                 5 #60Gln Val Arg Gly Arg Ser                 565 <210> SEQ ID NO 9<211> LENGTH: 1618 <212> TYPE: DNA<213> ORGANISM: Leishmania braziliensis <400> SEQUENCE: 9ccactctctc ggtcgtctgt ctcccacgcg cgcacgcagt tgatttccgc ct#tcttaaac     60gctctctttt tttttatttt tcacctgacc aaccgcacca cgtcggcctc ca#tcatgtcg    120cagcaagacc gagttgcccc acaggaccag gactcgttcc tcgacgacca gc#ccggcgtc    180cgcccgatcc cgtccttcga tgacatgccg ttgcaccaga accttctgcg cg#gcatctac    240tcgtacggct tcgagaaacc gtccagcatc cagcagcgcg ccatcgcccc ct#tcacgcgc    300ggcggcgaca tcatcgcgca ggcgcagtcc ggtaccggca agacgggcgc ct#tctccatc    360ggcctgctgc agcgcctgga cttccgccac aacctgatcc agggcctcgt gc#tctccccg    420acccgcgagc tggccctgca gacggcggag gtgatcagcc gcatcggcga gt#tcctgtcg    480aacagcgcga agttctgtga gacctttgtg ggtggcacgc gcgtgcagga tg#acctgcgc    540aagctgcagg ctggcgtcgt cgtcgccgtg gggacgccgg gccgcgtgtc cg#acgtgatc    600aagcgcggcg cgctgcgcac cgagtccctg cgcgtgctgg tgctcgacga gg#ctgatgag    660atgctgtctc agggcttcgc ggatcagatt tacgagatct tccgcttcct gc#cgaaggac    720atccaggtcg cgctcttctc cgccacgatg ccggaggagg tgctggagct ga#caaagaag    780ttcatgcgcg accccgtacg cattctcgtg aagcgcgaga gcctgacgct gg#agggcatc    840aagcagttct tcatcgccgt cgaggaggag cacaagctgg acacgctgat gg#acctgtac    900gagaccgtgt ccatcgcgca gtccgtcatc ttcgccaaca cccgccgcaa gg#tggactgg    960atcgccgaga agctgaatca gagcaaccac accgtcagca gcatgcacgc cg#agatgccc   1020aagagcgacc gcgagcgcgt catgaacacc ttccgcagcg gcagctcccg cg#tgctcgta   1080acgaccgacc tcgtggcccg cggcatcgac gtgcaccacg tgaacatcgt ca#tcaacttc   1140gacctgccga cgaacaagga gaactacctg caccgcattg gccgcggcgg cc#gctacggc   1200gtaaagggtg ttgccatcaa cttcgtgacg gagaaagacg tggagctgct gc#acgagatc   1260gaggggcact accacacgca gatcgatgag ctcccggtgg actttgccgc ct#acctcggc   1320gagtgagcgg gcccctgccc cccttccctg cccccctctc gcgacgagag aa#cgcacatc   1380gtaacacagc cacgcgaacg atagtaaggg cgtgcggcgg cgttcccctc ct#cctgccag   1440cggcccccct ccgcagcgct tctcttttga gaggggggca gggggaggcg ct#gcgcctgg   1500ctggatgtgt gcttgagctt gcattccgtc aagcaagtgc tttgttttaa tt#atgcgcgc   1560cgttttgttg ctcgtccctt tcgttggtgt tttttcggcc gaaacggcgt tt#aaagca     1618 210> SEQ ID NO 10 <211> LENGTH: 403 <212> TYPE: PRT<213> ORGANISM: Leishmania braziliensis <400> SEQUENCE: 10Met Ser Gln Gln Asp Arg Val Ala Pro Gln As #p Gln Asp Ser Phe Leu 1               5   #                10   #                15Asp Asp Gln Pro Gly Val Arg Pro Ile Pro Se #r Phe Asp Asp Met Pro            20       #            25       #            30Leu His Gln Asn Leu Leu Arg Gly Ile Tyr Se #r Tyr Gly Phe Glu Lys        35           #        40           #        45Pro Ser Ser Ile Gln Gln Arg Ala Ile Ala Pr #o Phe Thr Arg Gly Gly    50               #    55               #    60Asp Ile Ile Ala Gln Ala Gln Ser Gly Thr Gl #y Lys Thr Gly Ala Phe65                   #70                   #75                   #80Ser Ile Gly Leu Leu Gln Arg Leu Asp Phe Ar #g His Asn Leu Ile Gln                85   #                90   #                95Gly Leu Val Leu Ser Pro Thr Arg Glu Leu Al #a Leu Gln Thr Ala Glu            100       #           105       #           110Val Ile Ser Arg Ile Gly Glu Phe Leu Ser As #n Ser Ala Lys Phe Cys        115           #       120           #       125Glu Thr Phe Val Gly Gly Thr Arg Val Gln As #p Asp Leu Arg Lys Leu    130               #   135               #   140Gln Ala Gly Val Val Val Ala Val Gly Thr Pr #o Gly Arg Val Ser Asp145                 1 #50                 1 #55                 1 #60Val Ile Lys Arg Gly Ala Leu Arg Thr Glu Se #r Leu Arg Val Leu Val                165   #               170   #               175Leu Asp Glu Ala Asp Glu Met Leu Ser Gln Gl #y Phe Ala Asp Gln Ile            180       #           185       #           190Tyr Glu Ile Phe Arg Phe Leu Pro Lys Asp Il #e Gln Val Ala Leu Phe        195           #       200           #       205Ser Ala Thr Met Pro Glu Glu Val Leu Glu Le #u Thr Lys Lys Phe Met    210               #   215               #   220Arg Asp Pro Val Arg Ile Leu Val Lys Arg Gl #u Ser Leu Thr Leu Glu225                 2 #30                 2 #35                 2 #40Gly Ile Lys Gln Phe Phe Ile Ala Val Glu Gl #u Glu His Lys Leu Asp                245   #               250   #               255Thr Leu Met Asp Leu Tyr Glu Thr Val Ser Il #e Ala Gln Ser Val Ile            260       #           265       #           270Phe Ala Asn Thr Arg Arg Lys Val Asp Trp Il #e Ala Glu Lys Leu Asn        275           #       280           #       285Gln Ser Asn His Thr Val Ser Ser Met His Al #a Glu Met Pro Lys Ser    290               #   295               #   300Asp Arg Glu Arg Val Met Asn Thr Phe Arg Se #r Gly Ser Ser Arg Val305                 3 #10                 3 #15                 3 #20Leu Val Thr Thr Asp Leu Val Ala Arg Gly Il #e Asp Val His His Val                325   #               330   #               335Asn Ile Val Ile Asn Phe Asp Leu Pro Thr As #n Lys Glu Asn Tyr Leu            340       #           345       #           350His Arg Ile Gly Arg Gly Gly Arg Tyr Gly Va #l Lys Gly Val Ala Ile        355           #       360           #       365Asn Phe Val Thr Glu Lys Asp Val Glu Leu Le #u His Glu Ile Glu Gly    370               #   375               #   380His Tyr His Thr Gln Ile Asp Glu Leu Pro Va #l Asp Phe Ala Ala Tyr385                 3 #90                 3 #95                 4 #00Leu Gly Glu <210> SEQ ID NO 11 <211> LENGTH: 12 <212> TYPE: PRT<213> ORGANISM: Leishmania donovani <220> FEATURE:<221> NAME/KEY: VARIANT <222> LOCATION: (1)...(5)<223> OTHER INFORMATION: Xaa = any amino aci #d <221> NAME/KEY: VARIANT<222> LOCATION: (6)...(6) <223> OTHER INFORMATION: Xaa = Leu or Lys<221> NAME/KEY: VARIANT <222> LOCATION: (7)...(12)<223> OTHER INFORMATION: Xaa = any amino aci #d <400> SEQUENCE: 11Xaa Gln Xaa Pro Gln Xaa Val Phe Asp Glu Xa #a Xaa  1               5  #                10 <210> SEQ ID NO 12 <211> LENGTH: 26 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Sense PCR pri mer <221> NAME/KEY: modified_base<222> LOCATION: (1)...(26) <223> OTHER INFORMATION: I<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(26)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 12ggaattcccc ncagctngtn ttcgac           #                  #              26 <210> SEQ ID NO 13 <211> LENGTH: 5 <212> TYPE: PRT<213> ORGANISM: Leishmania donovani <400> SEQUENCE: 13Lys Val Phe Asp Glu  1               5 <210> SEQ ID NO 14<211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: PCR primer <400> SEQUENCE: 14ggatccatgg tcaagtccca ctacatctgc          #                  #           30 <210> SEQ ID NO 15 <211> LENGTH: 33 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: PCR primer <400> SEQUENCE: 15gaattcagac cggatagaaa taagccaatg aaa        #                  #         33 <210> SEQ ID NO 16 <211> LENGTH: 701 <212> TYPE: PRT<213> ORGANISM: Leishmania amozonensis <400> SEQUENCE: 16Met Thr Glu Thr Phe Ala Phe Gln Ala Glu Il #e Asn Gln Leu Met Ser 1               5   #                10   #                15Leu Ile Ile Asn Thr Phe Tyr Ser Asn Lys Gl #u Ile Phe Leu Arg Asp            20       #            25       #            30Val Ile Ser Asn Ala Ser Asp Ala Cys Asp Ly #s Ile Arg Tyr Gln Ser        35           #        40           #        45Leu Thr Asp Pro Ala Val Leu Gly Asp Ala Th #r Arg Leu Cys Val Arg    50               #    55               #    60Val Val Pro Asp Lys Glu Asn Lys Thr Leu Th #r Val Glu Asp Asn Gly65                   #70                   #75                   #80Ile Gly Met Thr Lys Ala Asp Leu Val Asn As #n Leu Gly Thr Ile Ala                85   #                90   #                95Arg Ser Gly Thr Lys Ala Phe Met Glu Ala Le #u Glu Ala Gly Ala Asp            100       #           105       #           110Met Ser Met Ile Gly Gln Phe Gly Val Gly Ph #e Tyr Ser Ala Tyr Leu        115           #       120           #       125Val Ala Asp Arg Val Thr Val Thr Ser Lys As #n Asn Ser Asp Glu Val    130               #   135               #   140Tyr Val Trp Glu Ser Ser Ala Gly Gly Thr Ph #e Thr Ile Thr Ser Ala145                 1 #50                 1 #55                 1 #60Pro Glu Ser Asp Met Lys Leu Pro Ala Arg Il #e Thr Leu His Leu Lys                165   #               170   #               175Glu Asp Gln Leu Glu Tyr Leu Glu Ala Arg Ar #g Leu Lys Glu Leu Ile            180       #           185       #           190Lys Lys His Ser Glu Phe Ile Gly Tyr Asp Il #e Glu Leu Met Val Glu        195           #       200           #       205Lys Thr Thr Glu Lys Glu Val Thr Asp Glu As #p Glu Glu Glu Ala Lys    210               #   215               #   220Lys Ala Asp Glu Asp Gly Glu Glu Pro Lys Va #l Glu Glu Val Thr Glu225                 2 #30                 2 #35                 2 #40Gly Glu Glu Asp Lys Lys Lys Lys Thr Lys Ly #s Val Lys Glu Val Thr                245   #               250   #               255Lys Glu Tyr Glu Val Gln Asn Lys His Lys Pr #o Leu Trp Thr Arg Asp            260       #           265       #           270Pro Lys Asp Val Thr Lys Glu Glu Tyr Ala Al #a Phe Tyr Lys Ala Ile        275           #       280           #       285Ser Asn Asp Trp Glu Asp Pro Pro Ala Thr Ly #s His Phe Ser Val Glu    290               #   295               #   300Gly Gln Leu Glu Phe Arg Ala Ile Met Phe Va #l Pro Lys Arg Ala Pro305                 3 #10                 3 #15                 3 #20Phe Asp Met Leu Glu Pro Asn Lys Lys Arg As #n Asn Ile Lys Leu Tyr                325   #               330   #               335Val Arg Arg Val Phe Ile Met Asp Asn Cys Gl #u Asp Leu Cys Pro Asp            340       #           345       #           350Trp Leu Gly Phe Val Lys Gly Val Val Asp Se #r Glu Asp Leu Pro Leu        355           #       360           #       365Asn Ile Ser Arg Glu Asn Leu Gln Gln Asn Ly #s Ile Leu Lys Val Ile    370               #   375               #   380Arg Lys Asn Ile Val Lys Lys Cys Leu Glu Me #t Phe Glu Glu Val Ala385                 3 #90                 3 #95                 4 #00Glu Asn Lys Glu Asp Tyr Lys Gln Phe Tyr Gl #u Gln Phe Gly Lys Asn                405   #               410   #               415Ile Lys Leu Gly Ile His Glu Asp Thr Ala As #n Arg Lys Lys Leu Met            420       #           425       #           430Glu Leu Leu Arg Phe Tyr Ser Thr Glu Ser Gl #y Glu Val Met Thr Thr        435           #       440           #       445Leu Lys Asp Tyr Val Thr Arg Met Lys Ala Gl #u Gln Asn Ser Ile Tyr    450               #   455               #   460Tyr Ile Thr Gly Asp Ser Lys Lys Lys Leu Gl #u Ser Ser Pro Phe Ile465                 4 #70                 4 #75                 4 #80Glu Gln Ala Lys Arg Arg Gly Phe Glu Val Le #u Phe Met Thr Glu Pro                485   #               490   #               495Tyr Asp Glu Tyr Val Met Gln Gln Val Lys As #p Phe Glu Asp Lys Lys            500       #           505       #           510Phe Ala Cys Leu Thr Lys Glu Gly Val His Ph #e Glu Glu Ser Glu Glu        515           #       520           #       525Glu Lys Lys Gln Arg Glu Glu Glu Lys Ala Th #r Cys Glu Lys Leu Cys    530               #   535               #   540Lys Thr Met Lys Glu Val Leu Gly Asp Lys Va #l Glu Lys Val Thr Val545                 5 #50                 5 #55                 5 #60Ser Glu Arg Leu Ser Thr Ser Pro Cys Ile Le #u Val Thr Ser Glu Phe                565   #               570   #               575Gly Trp Ser Ala His Met Glu Gln Met Met Ar #g Asn Gln Ala Leu Arg            580       #           585       #           590Asp Ser Ser Met Ala Gln Tyr Met Met Ser Ly #s Lys Thr Met Glu Leu        595           #       600           #       605Asn Pro Lys His Pro Ile Ile Lys Glu Leu Ar #g Arg Arg Val Glu Ala    610               #   615               #   620Asp Glu Asn Asp Lys Ala Val Lys Asp Leu Va #l Phe Leu Leu Phe Asp625                 6 #30                 6 #35                 6 #40Thr Ser Leu Leu Thr Ser Gly Phe Gln Leu Gl #u Asp Pro Thr Tyr Ala                645   #               650   #               655Glu Arg Ile Asn Arg Met Ile Lys Leu Gly Le #u Ser Leu Asp Glu Glu            660       #           665       #           670Glu Glu Glu Glu Ala Val Glu Ala Ala Val Al #a Glu Thr Ala Pro Ala        675           #       680           #       685Glu Val Thr Ala Gly Thr Ser Ser Met Glu Le #u Val Asp    690               #   695               #   700 <210> SEQ ID NO 17<211> LENGTH: 704 <212> TYPE: PRT <213> ORGANISM: T. Cruzi<400> SEQUENCE: 17 Met Thr Glu Thr Phe Ala Phe Gln Ala Glu Il#e Asn Gln Leu Met Ser  1               5   #                10  #                15 Leu Ile Ile Asn Thr Phe Tyr Ser Asn Lys Gl#u Ile Phe Leu Arg Glu             20       #            25      #            30 Leu Ile Ser Asn Ala Ser Asp Ala Cys Asp Ly#s Ile Arg Tyr Gln Ser         35           #        40          #        45 Leu Thr Asn Gln Ala Val Leu Gly Asp Glu Se#r His Leu Arg Ile Arg     50               #    55              #    60 Val Val Pro Asp Lys Ala Asn Lys Thr Leu Th#r Val Glu Asp Thr Gly 65                   #70                  #75                   #80 Ile Gly Met Thr Lys Ala Glu Leu Val Asn As#n Leu Gly Thr Ile Ala                 85   #                90  #                95 Arg Ser Gly Thr Lys Ala Phe Met Glu Ala Le#u Glu Ala Gly Gly Asp             100       #           105      #           110 Met Ser Met Ile Gly Gln Phe Gly Val Gly Ph#e Tyr Ser Ala Tyr Leu         115           #       120          #       125 Val Ala Asp Arg Val Thr Val Val Ser Lys As#n Asn Asp Asp Glu Ala     130               #   135              #   140 Tyr Thr Trp Glu Ser Ser Ala Gly Gly Thr Ph#e Thr Val Thr Pro Thr 145                 1 #50                 1#55                 1 #60 Pro Asp Cys Asp Leu Lys Arg Gly Thr Arg Il#e Val Leu His Leu Lys                 165   #               170  #               175 Glu Asp Gln Gln Glu Tyr Leu Glu Glu Arg Ar#g Leu Lys Asp Leu Ile             180       #           185      #           190 Lys Lys His Ser Glu Phe Ile Gly Tyr Asp Il#e Glu Leu Met Val Glu         195           #       200          #       205 Lys Ala Thr Glu Lys Glu Val Thr Asp Glu As#p Glu Asp Glu Ala Ala     210               #   215              #   220 Ala Thr Lys Asn Glu Glu Gly Glu Glu Pro Ly#s Val Glu Glu Val Lys 225                 2 #30                 2#35                 2 #40 Asp Asp Ala Glu Glu Gly Glu Lys Lys Lys Ly#s Thr Lys Lys Val Lys                 245   #               250  #               255 Glu Val Thr Gln Glu Phe Val Val Gln Asn Ly#s His Lys Pro Leu Trp             260       #           265      #           270 Thr Arg Asp Pro Lys Asp Val Thr Lys Glu Gl#u Tyr Ala Ala Phe Tyr         275           #       280          #       285 Lys Ala Ile Ser Asn Asp Trp Glu Glu Pro Le#u Ser Thr Lys His Phe     290               #   295              #   300 Ser Val Glu Gly Gln Leu Glu Phe Arg Ala Il#e Leu Phe Val Pro Lys 305                 3 #10                 3#15                 3 #20 Arg Ala Pro Phe Asp Met Phe Glu Pro Ser Ly#s Lys Arg Asn Asn Ile                 325   #               330  #               335 Lys Leu Tyr Val Arg Arg Val Phe Ile Met As#p Asn Cys Glu Asp Leu             340       #           345      #           350 Cys Pro Glu Trp Leu Ala Phe Val Arg Gly Va#l Val Asp Ser Glu Asp         355           #       360          #       365 Leu Pro Leu Asn Ile Ser Arg Glu Asn Leu Gl#n Gln Asn Lys Ile Leu     370               #   375              #   380 Lys Val Ile Arg Lys Asn Ile Val Lys Lys Al#a Leu Glu Leu Phe Glu 385                 3 #90                 3#95                 4 #00 Glu Ile Ala Glu Asn Lys Glu Asp Tyr Lys Ly#s Phe Tyr Glu Gln Phe                 405   #               410  #               415 Gly Lys Asn Val Lys Leu Gly Ile His Glu As#p Ser Ala Asn Arg Lys             420       #           425      #           430 Lys Leu Met Glu Leu Leu Arg Phe His Ser Se#r Glu Ser Gly Glu Asp         435           #       440          #       445 Met Thr Thr Leu Lys Asp Tyr Val Thr Arg Me#t Lys Glu Gly Gln Lys     450               #   455              #   460 Cys Ile Tyr Tyr Val Thr Gly Asp Ser Lys Ly#s Lys Leu Glu Thr Ser 465                 4 #70                 4#75                 4 #80 Pro Phe Ile Glu Gln Ala Arg Arg Arg Gly Ph#e Glu Val Leu Phe Met                 485   #               490  #               495 Thr Glu Pro Ile Asp Glu Tyr Val Met Gln Gl#n Val Lys Asp Phe Glu             500       #           505      #           510 Asp Lys Lys Phe Ala Cys Leu Thr Lys Glu Gl#y Val His Phe Glu Glu         515           #       520          #       525 Thr Glu Glu Glu Lys Lys Gln Arg Glu Glu Gl#u Lys Thr Ala Tyr Glu     530               #   535              #   540 Arg Leu Cys Lys Ala Met Lys Asp Val Leu Gl#y Asp Lys Val Glu Lys 545                 5 #50                 5#55                 5 #60 Val Val Val Ser Glu Arg Leu Ala Thr Ser Pr#o Cys Ile Leu Val Thr                 565   #               570  #               575 Ser Glu Phe Gly Trp Ser Ala His Met Glu Gl#n Ile Met Arg Asn Gln             580       #           585      #           590 Ala Leu Arg Asp Ser Ser Met Ser Ala Tyr Me#t Met Ser Lys Lys Thr         595           #       600          #       605 Met Glu Ile Asn Pro Ala His Pro Ile Val Ly#s Glu Leu Lys Arg Arg     610               #   615              #   620 Val Glu Ala Asp Glu Asn Asp Lys Ala Val Ly#s Asp Leu Val Tyr Leu 625                 6 #30                 6#35                 6 #40 Leu Phe Asp Thr Ala Leu Leu Thr Ser Gly Ph#e Thr Leu Asp Asp Pro                 645   #               650  #               655 Thr Ser Tyr Ala Glu Arg Ile His Arg Met Il#e Lys Leu Gly Leu Ser             660       #           665      #           670 Leu Asp Asp Glu Asp Asn Gly Asn Glu Glu Al#a Glu Pro Ala Ala Ala         675           #       680          #       685 Val Pro Ala Glu Pro Val Ala Gly Thr Ser Se#r Met Glu Gln Val Asp     690               #   695              #   700 <210> SEQ ID NO 18 <211> LENGTH: 732 <212> TYPE: PRT<213> ORGANISM: Homo sapien <400> SEQUENCE: 18Met Pro Glu Glu Thr Gln Thr Gln Asp Gln Pr #o Met Glu Glu Glu Glu 1               5   #                10   #                15Val Glu Thr Phe Ala Phe Gln Ala Glu Ile Al #a Gln Leu Met Ser Leu            20       #            25       #            30Ile Ile Asn Thr Phe Tyr Ser Asn Lys Glu Il #e Phe Leu Arg Glu Leu        35           #        40           #        45Ile Ser Asn Ser Ser Asp Ala Leu Asp Lys Il #e Arg Tyr Glu Ser Leu    50               #    55               #    60Thr Asp Pro Ser Lys Leu Asp Ser Gly Lys Gl #u Leu His Ile Asn Leu65                   #70                   #75                   #80Ile Pro Asn Lys Gln Asp Arg Ala Leu Thr Il #e Val Asp Thr Gly Ile                85   #                90   #                95Gly Met Thr Lys Ala Asp Leu Ile Asn Asn Le #u Gly Thr Ile Ala Lys            100       #           105       #           110Ser Gly Thr Lys Ala Phe Met Glu Ala Leu Gl #n Ala Gly Ala Asp Ile        115           #       120           #       125Ser Met Ile Gly Gln Phe Gly Val Gly Phe Ty #r Ser Ala Tyr Leu Val    130               #   135               #   140Ala Glu Lys Val Thr Val Ile Thr Lys His As #n Asp Asp Glu Gln Tyr145                 1 #50                 1 #55                 1 #60Ala Trp Glu Ser Ser Ala Gly Gly Ser Phe Th #r Val Arg Thr Asp Thr                165   #               170   #               175Gly Glu Pro Met Gly Arg Gly Thr Lys Val Il #e Leu His Leu Lys Glu            180       #           185       #           190Asp Gln Thr Glu Tyr Leu Glu Glu Arg Arg Il #e Lys Glu Ile Val Lys        195           #       200           #       205Lys His Ser Gln Phe Ile Gly Tyr Pro Ile Th #r Leu Phe Val Glu Lys    210               #   215               #   220Glu Arg Asp Lys Glu Val Ser Asp Asp Glu Al #a Glu Glu Lys Glu Asp225                 2 #30                 2 #35                 2 #40Lys Glu Glu Glu Lys Glu Lys Glu Glu Lys Gl #u Ser Glu Asp Lys Pro                245   #               250   #               255Glu Ile Glu Asp Val Gly Ser Asp Glu Glu As #p Glu Lys Lys Asp Gly            260       #           265       #           270Asp Lys Lys Lys Lys Lys Lys Ile Lys Glu Ly #s Tyr Ile Asp Lys Glu        275           #       280           #       285Glu Leu Asn Lys Thr Lys Pro Ile Trp Thr Ar #g Asn Pro Asp Asp Ile    290               #   295               #   300Thr Asn Glu Glu Tyr Gly Glu Phe Tyr Lys Se #r Leu Thr Asn Asp Trp305                 3 #10                 3 #15                 3 #20Glu Asp His Leu Ala Val Lys His Phe Ser Va #l Glu Gly Gln Leu Glu                325   #               330   #               335Phe Arg Ala Leu Leu Phe Val Pro Arg Arg Al #a Pro Phe Asp Leu Phe            340       #           345       #           350Glu Asn Arg Lys Lys Lys Asn Asn Ile Lys Le #u Tyr Val Arg Arg Val        355           #       360           #       365Phe Ile Met Asp Asn Cys Glu Glu Leu Ile Pr #o Glu Tyr Leu Asn Phe    370               #   375               #   380Ile Arg Gly Val Val Asp Ser Glu Asp Leu Pr #o Leu Asn Ile Ser Arg385                 3 #90                 3 #95                 4 #00Glu Met Leu Gln Gln Ser Lys Ile Leu Lys Va #l Ile Arg Lys Asn Leu                405   #               410   #               415Val Lys Lys Cys Leu Glu Leu Phe Thr Glu Le #u Ala Glu Asp Lys Glu            420       #           425       #           430Asn Tyr Lys Lys Phe Tyr Glu Gln Phe Ser Ly #s Asn Ile Lys Leu Gly        435           #       440           #       445Ile His Glu Asp Ser Gln Asn Arg Lys Lys Le #u Ser Glu Leu Leu Arg    450               #   455               #   460Tyr Tyr Thr Ser Ala Ser Gly Asp Glu Met Va #l Ser Leu Lys Asp Tyr465                 4 #70                 4 #75                 4 #80Cys Thr Arg Met Lys Glu Asn Gln Lys His Il #e Tyr Tyr Ile Thr Gly                485   #               490   #               495Glu Thr Lys Asp Gln Val Ala Asn Ser Ala Ph #e Val Glu Arg Leu Arg            500       #           505       #           510Lys His Gly Leu Glu Val Ile Tyr Met Ile Gl #u Pro Ile Asp Glu Tyr        515           #       520           #       525Cys Val Gln Gln Leu Lys Glu Phe Glu Gly Ly #s Thr Leu Val Ser Val    530               #   535               #   540Thr Lys Glu Gly Leu Glu Leu Pro Glu Asp Gl #u Glu Glu Lys Lys Lys545                 5 #50                 5 #55                 5 #60Gln Glu Glu Lys Lys Thr Lys Phe Glu Asn Le #u Cys Lys Ile Met Lys                565   #               570   #               575Asp Ile Leu Glu Lys Lys Val Glu Lys Val Va #l Val Ser Asn Arg Leu            580       #           585       #           590Val Thr Ser Pro Cys Cys Leu Val Thr Ser Th #r Tyr Gly Trp Thr Ala        595           #       600           #       605Asn Met Glu Arg Ile Met Lys Ala Gln Ala Le #u Arg Asp Asn Ser Thr    610               #   615               #   620Met Gly Tyr Met Ala Ala Lys Lys His Leu Gl #u Ile Asn Pro Asp His625                 6 #30                 6 #35                 6 #40Ser Ile Ile Glu Thr Leu Arg Gln Lys Ala Gl #u Ala Asp Lys Asn Asp                645   #               650   #               655Lys Ser Val Lys Asp Leu Val Ile Leu Leu Ty #r Glu Thr Ala Leu Leu            660       #           665       #           670Ser Ser Gly Phe Ser Leu Glu Asp Pro Gln Th #r His Ala Asn Arg Ile        675           #       680           #       685Tyr Arg Met Ile Lys Leu Gly Leu Gly Ile As #p Glu Asp Asp Pro Thr    690               #   695               #   700Ala Asp Asp Thr Ser Ala Ala Val Thr Glu Gl #u Met Pro Pro Leu Glu705                 7 #10                 7 #15                 7 #20Gly Asp Asp Asp Thr Ser Arg Met Glu Glu Va #l Asp                   #    725              #    730 <210> SEQ ID NO 19 <211> LENGTH: 1019<212> TYPE: DNA <213> ORGANISM: Leishmania major <400> SEQUENCE: 19gaattcggca cgaggtttct gtactttatt gcttccagcc tttattcact ct#tcgatttc     60ctctaacacc atgtcctccg agcgcacctt tattgccgtc aagccggacg gc#gtgcagcg    120cggcctcgtt ggcgagatca tcgcccgctt cgagcgcaag ggctacaagc tc#gtcgcctt    180gaagatactg cagccgacga cggagcaggc ccagggtcac tataaggacc tt#tgctccaa    240gccgtttttc ccggcccttg tgaagtactt ctcctctggc ccgatcgtgt gt#atggtgtg    300ggagggtaag aacgtggtga agagcggccg cgtgctgctc ggcgcgacga ac#ccggccga    360ctcacagccc ggcacgatcc gtggcgactt tgccgtggat gtgggccgca ac#gtgtgcca    420cgggtccgac tctgtggaga gcgcggagcg cgagatcgcc ttttggttca ag#gcggatga    480gatcgcgagc tggacgtcgc actccgtgtc ccagatctat gagtaacggt ga#ttgcggac    540acgctttgag gacgtagctg tacccccaat gaattcttct ctgaaaacca ca#tcataagc    600ctcttaagag gttatttttc ttgatcgatg cccggtggtg accagcacca tt#cctttatc    660ggattcactc acactcctag cgaatcatgt agtgcggtga gagtgggctc tg#gaggagac    720tgttgtgtag ccatggcttc aggagagaaa acaaaataca aggaaaggca at#atgtaact    780atggggttcc cttttttact atgcaaagtt tttataactc ctgatcggca aa#aacaacaa    840caaccgccat acaccaagag caaatgcttt cttctgcgga ctgtgcttct gt#tttttttt    900atgaaggagt gactcgcgcg atgaaaagtg tgtgcgtggg agatgtattt cc#tttttttg    960ttcatagtgg cgacagctca ctgttgacga tgacaaaaaa aaaaaaaaaa aa#actcgag    1019 <210> SEQ ID NO 20 <211> LENGTH: 151 <212> TYPE: PRT<213> ORGANISM: Leishmania major <400> SEQUENCE: 20Met Ser Ser Glu Arg Thr Phe Ile Ala Val Ly #s Pro Asp Gly Val Gln 1               5   #                10   #                15Arg Gly Leu Val Gly Glu Ile Ile Ala Arg Ph #e Glu Arg Lys Gly Tyr            20       #            25       #            30Lys Leu Val Ala Leu Lys Ile Leu Gln Pro Th #r Thr Glu Gln Ala Gln        35           #        40           #        45Gly His Tyr Lys Asp Leu Cys Ser Lys Pro Ph #e Phe Pro Ala Leu Val    50               #    55               #    60Lys Tyr Phe Ser Ser Gly Pro Ile Val Cys Me #t Val Trp Glu Gly Lys65                   #70                   #75                   #80Asn Val Val Lys Ser Gly Arg Val Leu Leu Gl #y Ala Thr Asn Pro Ala                85   #                90   #                95Asp Ser Gln Pro Gly Thr Ile Arg Gly Asp Ph #e Ala Val Asp Val Gly            100       #           105       #           110Arg Asn Val Cys His Gly Ser Asp Ser Val Gl #u Ser Ala Glu Arg Glu        115           #       120           #       125Ile Ala Phe Trp Phe Lys Ala Asp Glu Ile Al #a Ser Trp Thr Ser His    130               #   135               #   140Ser Val Ser Gln Ile Tyr Glu 145                 1 #50 <210> SEQ ID NO 21<211> LENGTH: 1523 <212> TYPE: DNA <213> ORGANISM: Leishmania major<400> SEQUENCE: 21gaattcggca cgagtgctgc ccgacatgac atgctcgctg accggacttc ag#tgcacaga     60cccgaactgc aagacctgca caacttacgg tcagtgcaca gactgcaacg ac#ggctacgg    120tctcacctcc tccagcgttt gcgtgcgctg cagtgtagcg ggctgcaaga gc#tgccccgt    180cgacgctaac gtctgcaaag tgtgtctcgg cggcagcgag ccgatcaaca at#atgtgccc    240ctgcaccgac cccaactgcg ccagctgccc cagcgacgct ggcacgtgca ct#cagtgcgc    300gaacggctac ggtctcgtgg acggcgcctg tgtgagatgc caggagccca ac#tgcttcag    360ctgcgacagc gacgcgaata agtgcacaca atgtgcgccg aactactacc tc#accccgct    420cttgacctgc tccccggtgg cctgcaacat cgagcactgc atgcagtgcg ac#ccacagac    480gccgtcgcgc tgccaggagt gcgtgtcccc ctacgtggtt gacagctacg ac#ggcctctg    540caggctctcc gatgcctgct ccgtgcccaa ctgcaagaag tgcgagaccg gt#acctccag    600gctctgcgcc gagtgcgaca ccggctacag tctctccgcc gacgcgacga gc#tgcagcag    660tccaaccacg cagccgtgcg aggtggagca ctgcaacaca tgtgtgaacg gc#gatagcac    720ccgctgtgcc tactgcaaca ccggctacta cgtctccgat ggcaagtgca ag#gccatgca    780gggctgctac gtgtcgaact gcgcgcagtg catgctgctt gacagcacca ag#tgctccac    840gtgcgtgaaa gggtacctgc tcacgtcgtc ctacagttgc gtctcgcaga aa#gtcatcaa    900cagtgcggcc gcgccctact ctctgtgggt ggccgccgcc gtgctcctca cc#tcttttgc    960catgcaccta gcatagtgcg cagcggcatg cgaacaaccc cactctcatt ct#ccaacatg   1020tgcatacaca cacacacaga cagcggggca gcaccccctc cccacacaca ca#cacgcact   1080tcccccttgt cttgttcttc tttcctcgtt cgcatttctt tctctcgtgc gc#tggcgccg   1140gcctcctgca cgtcgctccc ctccccctaa cctctattct ctctctctct ct#ctctcgcc   1200ggcatcattg cttcttaccc ttttctgatc cttgctcgcg tgggcggaca ct#gccacagt   1260cccacagcgc agacacacgt gtttaaacgg cgcaggcatc cctccctatc ac#ttcatttc   1320tcctaaagcc actcaccaag tcgcacaccg ccctccccca tcggccgccc tt#ccgggcgc   1380agctgtgcgg aatgggtgtg tgctcgacct cgttcctggc agctcactcg ca#tgtgtaca   1440gccactccaa ccacgaaagc tctcttctgc gcacataaaa aaaaaaaaaa aa#aaaaaact   1500 cgaggggggg cccggtaccc aaa           #                   #              1523 <210> SEQ ID NO 22<211> LENGTH: 320 <212> TYPE: PRT <213> ORGANISM: Leishmania major<400> SEQUENCE: 22 Val Leu Pro Asp Met Thr Cys Ser Leu Thr Gl#y Leu Gln Cys Thr Asp  1               5   #                10  #                15 Pro Asn Cys Lys Thr Cys Thr Thr Tyr Gly Gl#n Cys Thr Asp Cys Asn             20       #            25      #            30 Asp Gly Tyr Gly Leu Thr Ser Ser Ser Val Cy#s Val Arg Cys Ser Val         35           #        40          #        45 Ala Gly Cys Lys Ser Cys Pro Val Asp Ala As#n Val Cys Lys Val Cys     50               #    55              #    60 Leu Gly Gly Ser Glu Pro Ile Asn Asn Met Cy#s Pro Cys Thr Asp Pro 65                   #70                  #75                   #80 Asn Cys Ala Ser Cys Pro Ser Asp Ala Gly Th#r Cys Thr Gln Cys Ala                 85   #                90  #                95 Asn Gly Tyr Gly Leu Val Asp Gly Ala Cys Va#l Arg Cys Gln Glu Pro             100       #           105      #           110 Asn Cys Phe Ser Cys Asp Ser Asp Ala Asn Ly#s Cys Thr Gln Cys Ala         115           #       120          #       125 Pro Asn Tyr Tyr Leu Thr Pro Leu Leu Thr Cy#s Ser Pro Val Ala Cys     130               #   135              #   140 Asn Ile Glu His Cys Met Gln Cys Asp Pro Gl#n Thr Pro Ser Arg Cys 145                 1 #50                 1#55                 1 #60 Gln Glu Cys Val Ser Pro Tyr Val Val Asp Se#r Tyr Asp Gly Leu Cys                 165   #               170  #               175 Arg Leu Ser Asp Ala Cys Ser Val Pro Asn Cy#s Lys Lys Cys Glu Thr             180       #           185      #           190 Gly Thr Ser Arg Leu Cys Ala Glu Cys Asp Th#r Gly Tyr Ser Leu Ser         195           #       200          #       205 Ala Asp Ala Thr Ser Cys Ser Ser Pro Thr Th#r Gln Pro Cys Glu Val     210               #   215              #   220 Glu His Cys Asn Thr Cys Val Asn Gly Asp Se#r Thr Arg Cys Ala Tyr 225                 2 #30                 2#35                 2 #40 Cys Asn Thr Gly Tyr Tyr Val Ser Asp Gly Ly#s Cys Lys Ala Met Gln                 245   #               250  #               255 Gly Cys Tyr Val Ser Asn Cys Ala Gln Cys Me#t Leu Leu Asp Ser Thr             260       #           265      #           270 Lys Cys Ser Thr Cys Val Lys Gly Tyr Leu Le#u Thr Ser Ser Tyr Ser         275           #       280          #       285 Cys Val Ser Gln Lys Val Ile Asn Ser Ala Al#a Ala Pro Tyr Ser Leu     290               #   295              #   300 Trp Val Ala Ala Ala Val Leu Leu Thr Ser Ph#e Ala Met His Leu Ala 305                 3 #10                 3#15                 3 #20 <210> SEQ ID NO 23 <211> LENGTH: 797<212> TYPE: DNA <213> ORGANISM: Leishmania major <400> SEQUENCE: 23ctgtacttta ttgccaccag ccagccatgt cctgcggtaa cgccaagatc aa#ctctcccg     60cgccgtcctt cgaggaggtg gcgctcatgc ccaacggcag cttcaagaag at#cagcctct    120cctcctacaa gggcaagtgg gtcgtgctct tcttctaccc gctcgacttt ag#cttcgtgt    180gcccgacaga ggtcatcgcg ttctccgaca gcgtgagtcg cttcaacgag ct#caactgcg    240aggtcctcgc gtgctcgata gacagcgagt acgcgcacct gcagtggacg ct#gcaggacc    300gcaagaaggg cggcctcggg accatggcga tcccaatgct agccgacaag ac#caagagca    360tcgctcgttc ctacggcgtg ctggaggaga gccagggcgt ggcctaccgc gg#tctcttca    420tcatcgaccc ccatggcatg ctgcgtcaga tcaccgtcaa tgacatgccg gt#gggccgca    480gcgtggagga ggttctacgc ctgctggagg cttttcagtt cgtggagaag ca#cggcgagg    540tgtgccccgc gaactggaag aagggcgccc ccacgatgaa gccggaaccg aa#tgcgtctg    600tcgagggata cttcagcaag cagtaaacct gtgagcgtcg caggagtcag tg#tgacctca    660cccgcctctg ccagtgggtg cgagagggcg tgagggattg tgggaaggct gt#tggatatg    720atgcagacag cgatgaatgc aactcccaca cactggccct cctcagccct ct#ccacacag    780 acacacgcac gcatgtg              #                  #                   #  797 <210> SEQ ID NO 24 <211> LENGTH: 199<212> TYPE: PRT <213> ORGANISM: Leishmania major <400> SEQUENCE: 24Met Ser Cys Gly Asn Ala Lys Ile Asn Ser Pr #o Ala Pro Ser Phe Glu 1               5   #                10   #                15Glu Val Ala Leu Met Pro Asn Gly Ser Phe Ly #s Lys Ile Ser Leu Ser            20       #            25       #            30Ser Tyr Lys Gly Lys Trp Val Val Leu Phe Ph #e Tyr Pro Leu Asp Phe        35           #        40           #        45Ser Phe Val Cys Pro Thr Glu Val Ile Ala Ph #e Ser Asp Ser Val Ser    50               #    55               #    60Arg Phe Asn Glu Leu Asn Cys Glu Val Leu Al #a Cys Ser Ile Asp Ser65                   #70                   #75                   #80Glu Tyr Ala His Leu Gln Trp Thr Leu Gln As #p Arg Lys Lys Gly Gly                85   #                90   #                95Leu Gly Thr Met Ala Ile Pro Met Leu Ala As #p Lys Thr Lys Ser Ile            100       #           105       #           110Ala Arg Ser Tyr Gly Val Leu Glu Glu Ser Gl #n Gly Val Ala Tyr Arg        115           #       120           #       125Gly Leu Phe Ile Ile Asp Pro His Gly Met Le #u Arg Gln Ile Thr Val    130               #   135               #   140Asn Asp Met Pro Val Gly Arg Ser Val Glu Gl #u Val Leu Arg Leu Leu145                 1 #50                 1 #55                 1 #60Glu Ala Phe Gln Phe Val Glu Lys His Gly Gl #u Val Cys Pro Ala Asn                165   #               170   #               175Trp Lys Lys Gly Ala Pro Thr Met Lys Pro Gl #u Pro Asn Ala Ser Val            180       #           185       #           190Glu Gly Tyr Phe Ser Lys Gln         195 <210> SEQ ID NO 25<211> LENGTH: 637 <212> TYPE: DNA <213> ORGANISM: Leishmania tropica<400> SEQUENCE: 25ttacatatgc atcaccacca ccaccacatg tcctgcggta acgccaagat ca#actctccc     60gcgccgccct tcgaggagat ggcgctcatg cccaacggca gcttcaagaa ga#tcagcctc    120tccgcctaca agggcaagtg ggtcgtgctc ttcttctacc cgctcgactt ca#ccttcgtg    180tgcccgacag agatcatcgc gttctccgac aacgtgagtc gcttcaacga gc#tcaactgc    240gaggtcctcg cgtgctcgat ggacagcgag tacgcgcacc tgcagtggac gc#tgcaggac    300cgcaagaagg gcggcctcgg ggccatggcg atcccaatgc tggccgacaa ga#ctaagagc    360atcgctcgtt cctacggcgt gctggaggag agccagggcg tggcctaccg cg#gtctcttc    420atcatcgacc cccgtggcat ggtgcgtcag atcaccgtca acgacatgcc gg#tgggccgc    480aacgtggagg aggctctgcg cctgctggag gctttgcagt tcgtggagaa gc#acggcgag    540gtgtgccccg cgaactggaa gaagggcgcc cccacgatga agccggaacc ga#aggcgtct    600 gtcgagggat acttcagcaa gcagtaagaa ttccatg      #                   #     637 <210> SEQ ID NO 26 <211> LENGTH: 206<212> TYPE: PRT <213> ORGANISM: Leishmania tropica <400> SEQUENCE: 26Met His His His His His His Met Ser Cys Gl #y Asn Ala Lys Ile Asn 1               5   #                10   #                15Ser Pro Ala Pro Pro Phe Glu Glu Met Ala Le #u Met Pro Asn Gly Ser            20       #            25       #            30Phe Lys Lys Ile Ser Leu Ser Ala Tyr Lys Gl #y Lys Trp Val Val Leu        35           #        40           #        45Phe Phe Tyr Pro Leu Asp Phe Thr Phe Val Cy #s Pro Thr Glu Ile Ile    50               #    55               #    60Ala Phe Ser Asp Asn Val Ser Arg Phe Asn Gl #u Leu Asn Cys Glu Val65                   #70                   #75                   #80Leu Ala Cys Ser Met Asp Ser Glu Tyr Ala Hi #s Leu Gln Trp Thr Leu                85   #                90   #                95Gln Asp Arg Lys Lys Gly Gly Leu Gly Ala Me #t Ala Ile Pro Met Leu            100       #           105       #           110Ala Asp Lys Thr Lys Ser Ile Ala Arg Ser Ty #r Gly Val Leu Glu Glu        115           #       120           #       125Ser Gln Gly Val Ala Tyr Arg Gly Leu Phe Il #e Ile Asp Pro Arg Gly    130               #   135               #   140Met Val Arg Gln Ile Thr Val Asn Asp Met Pr #o Val Gly Arg Asn Val145                 1 #50                 1 #55                 1 #60Glu Glu Ala Leu Arg Leu Leu Glu Ala Leu Gl #n Phe Val Glu Lys His                165   #               170   #               175Gly Glu Val Cys Pro Ala Asn Trp Lys Lys Gl #y Ala Pro Thr Met Lys            180       #           185       #           190Pro Glu Pro Lys Ala Ser Val Glu Gly Tyr Ph #e Ser Lys Gln        195           #       200           #       205<210> SEQ ID NO 27 <211> LENGTH: 51 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: PCR primer <400> SEQUENCE: 27 caattacata tgcatcacca tcaccatcac atgtcctgcg gtaacgccaa #g              51 <210> SEQ ID NO 28 <211> LENGTH: 31 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: PCR primer <400> SEQUENCE: 28catggaattc ttactgcttg ctgaagtatc c         #                  #          31 <210> SEQ ID NO 29 <211> LENGTH: 520 <212> TYPE: DNA<213> ORGANISM: Leishmania major <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(520)<223> OTHER INFORMATION: n = A, T, C or  #G <400> SEQUENCE: 29ggcacgagcc cttgcctaca tttgctcgcc gatattcgcg gggagttctt ca#atttgcgt     60cgcgtagaac tgctcaatgt cgcgcaacaa gcgcagctcg tcgtggcgca cg#aaggtgat    120ggccagtcca gtgcggccca tgcggccagt gcggccgatg cggtgaatgt ac#tgctcacg    180cgcgagcggc aaatcgtagc tgaggacgag cgagacgcgc tccacatcaa tg#ccacgcgc    240ccacaggtcc gttgtaatga ncacgcggct gtgtccatta cggaatgccg ca#taatctcg    300tcgcgctccg cctggggcat gtcgccgtgc atggcggaca cagcgaaatt ct#cgcgcgtc    360atcttcttgg caagctgctc cacctttttg cgggtgttgc anaaaaccac ng#cgtgggcg    420atcgttaagc tgtcgtacaa actccatcaa gaaatcgaat ttgtttttct ct#tcgtcnac    480 nganacaaan tactgtttaa cgctntccac ggtgatctca     #                   #   520 <210> SEQ ID NO 30 <211> LENGTH: 600<212> TYPE: DNA <213> ORGANISM: Leishmania major <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(600)<223> OTHER INFORMATION: n = A, T, C or  #G <400> SEQUENCE: 30ggcacaaggt tttcgggtta tcttcacgca tggtggagcg cagatgggtg aa#gtaaatac     60gcggaccgaa ctgcttgatc atatcaacca gatcgttgtc agcacgcacg cc#gtangaac    120cggtgcacat ggtaaaaccg tntgccatgc tgtttacggt atcaaccatc ca#ctgcatat    180cttcaatggt ggaaacaatg cgcggcaggc cgaggatccg gcgcggctca tc#atnnagnt    240natnaaccan tcgcacgtct anttctgcac taaactacaa ntatcggtna ca#tatnataa    300ggccnatttt cggtccagga ntatgtnctn tcaaaatgcc ncgttannca ct#cttaaatg    360tctcangngn aaantngttc taaagggtgt ccaaaanntn nttaccnttc cc#cncttact    420tcaananctc ctcnaattcc cnggcccttn gacnannatt tnctattaaa an#atanaann    480ttcaaattna ttcccnacct nccntnncca aanntancna ataatcannc cc#ctntcann    540anntcccanc ttaccctccn ntngnngggn nnnccnattn ccccaanccc nc#nctaaata    600 210> SEQ ID NO 31 <211> LENGTH: 600 <212> TYPE: DNA<213> ORGANISM: Leishmania major <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(600)<223> OTHER INFORMATION: n = A, T, C or  #G <400> SEQUENCE: 31ggcacgagcc tcagtggagc tcaatgaaga tattgcagta tcttactctg ga#tggcactc     60aggtctccgg cacgctgccg ccccagtgga gcgcgatggc atcggtgcga at#tcttaacc    120tgnagggtac tgaggtctct ggtacgctgc cgcctgagtg gatatcnatg an#caggctgc    180aaactctgaa tctgcggcgc acgaaantat ccggcactct gccgcccgaa tg#ganttcta    240tgaacagcct ggagtacttt cacctttatc ttactcaggt ctccggcacg ct#gccgcccg    300agtggagtgg gatgtcnaag gccgcatact tctggctgga atactgcgac ct#gtccggca    360ntctgccgcc cnagtggtcg tcnatgccaa agctgcgcgg tatctcactg an#cggcaaca    420aattcttgcg ngtgtntncc ngactcntgg gattcagaaa ggtggtcctt gt#tgttgggc    480atcnaaggan caaaccccaa ngggcccncn aattgcttgg gcntgcttaa gg#anttgcac    540naaccaacnc cnccaaaaac cccccccacc ncnaaannac nancccccac tt#aanncccn    600 <210> SEQ ID NO 32 <211> LENGTH: 600 <212> TYPE: DNA<213> ORGANISM: Leishmania major <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(600)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 32ngcacgagaa gcgcaactgg cgcatcgcat ctgtgactat ctgcctgaac ag#gggcaatn     60gtttgttggt aacagcctgg tggtacgtct gattgatncg cttncgcaan tt#ccggcagg    120ttacccggtg tacancaacc gtggggccan cggtatcnac nggctgcttt cg#accgccgc    180cggngttcan cgggcaancg gcaaaccgac gctggcgatt gtgggcgatc tc#tccgcact    240ttacgatctc aacgcnctgg cgttattgcg tcaggtttct gcgccgctgg ta#ttaattgt    300ggtgaacaac aacggcnggg caaaattttc tcgctgttgc caacgccccc aa#agcnagcg    360tgaagcgttt ctatctgatg ccgcaaaacg tccattttga aacacgccgc cn#cccatgtt    420tcganctgaa aatatcatcg tccgcaaaac tggcangaaa cttngaaaac cg#cattttgc    480cgacnccctg gcncacgccc aacccaccca ccggttgatt gaaaatggtg gg#ttaacgaa    540nccnnatggg tgccccaaan cncnnccanc caaatttctg ggcccaggtt aa#ancccttt    600 <210> SEQ ID NO 33 <211> LENGTH: 600 <212> TYPE: DNA<213> ORGANISM: Leishmania major <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(600)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 33acgatgacca tgccccgaag gaggatggcc atgcgccgaa gaacgatgac ca#tgccccga     60aggaggatgg ccatgcgccg aagaacgatg accatgcccc gaaggaggat gg#ccatgcgc    120cgaagaacga cggggatgtg cagaanaaga gcgaagatgg agacaacgtg gg#agagggag    180gcaagggcaa tgaggatggt aacgatgatc agccgaagga gcacgctgcc gg#caactagt    240gggctgcgtc cgggcttgtg tgcganccgt gctctgcacc ccgccgctcg tg#catcctcg    300catgtggact gcgtgtgtct ctcccgcttt gtctctctcc cccacacagt gg#ctgatgcc    360tgcacggggt tgctgtggct gcacctcctg accactgcca gctttcttgg ct#tgcctccc    420ctctgcgcct ccgctcgtgc cgctcgtgcc gaattcgata tcaagcttat cg#ataccgtc    480nacctcgaag gggggcccgg ttacccattc gccctatant gagtcntatt ac#aattcctg    540gcgtcgtttt acacgtcgtg actgggaaaa accctggcgt tccccactta tc#gccttgca    600 <210> SEQ ID NO 34 <211> LENGTH: 516 <212> TYPE: DNA<213> ORGANISM: Leishmania major <400> SEQUENCE: 34agctgcagca gcgcctagac accgccacgc agcagcgcgc cgagctggag gc#acgggtgg     60cacggctggc cgcggaccgc gacgaggcgc gccagcagct ggccgcgaac gc#cgaggagc    120tgcagcagcg cctagacacc gccacgcagc agcgcgccga gctggaggca cg#ggtggcac    180ggctggccgc ggacggcgac gaggcccgcc agcagctggc cgcgaacgcc ga#ggagctgc    240agcagcgcct agacaccgcc acgcagcagc gcgccgagct ggaggcacag gt#ggcacggc    300tggccgcgaa cgccgaggag ctgcagcagc gcctagacac cgccacgcag ca#gcgcgccg    360agctggaggc acgggtggca cggctggccg cggaccgcga cgaggcgcgc ca#gcagctgg    420ccgcgaacgc cgaggagctg cagcagcgcc tagacaccgc cacgcagcag cg#cgccgagc    480 tggargcaca ggtggcacgg ctggccgcga amgccg      #                   #      516 <210> SEQ ID NO 35 <211> LENGTH: 822<212> TYPE: DNA <213> ORGANISM: Leishmania major <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(822)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 35ggcacganag atcttcgtga agacgctgac cggcaanacg atcgcgctgg ag#gtggagcc     60gagcgacacg atcgagaacg tgaaggccaa gatccaggac aaggagggca tc#ccgccgga    120ccagcagcgc ctgatcttcg ccggcaagca gctggaggan ggccgcacgc tc#tcggacta    180caacatccag aaggagtcca cgctgcacct ggtgctgcgc ctgcgcggcg gc#atgcanat    240cttcgtgaaa acgctnaccg gcaanacaat cgcgctggaa gtggagccga ac#gaccnatc    300gaaaacgtga aggccnanat ccangacaag gaaggcntcc cgccgganca gc#acgcctga    360tcttccnccg gcaaccactt gangaagggc ncacgctctc ngactacnac at#ccanaaag    420gattccnccc tgcaccttgt tgcttgcncc ttgctcgggg ggcatgccna at#cttccttn    480aaaacctcaa ccggcaanaa caatcccccn cngaagttgg aacccaacca nc#ccattcna    540aaactttaaa ggccnnnatt ccngaacaan gaagggcttc ccccccggac cn#ncaancnc    600cctgattntt cccccggnnn ncantttgga angaagggcc ccnccctccn cc#gaattncn    660acntcccnaa anggattccc cccctnccct tgntttttgc gccnnnnnnc gg#cnncntnc    720cnaaattccg nccnaaggnc cccantanan cnactttccc nttccccccc nn#nnttttgc    780 ntaaantttt tncccccnna aanntcccnt ttncnanttn an    #                   # 822 <210> SEQ ID NO 36 <211> LENGTH: 146<212> TYPE: PRT <213> ORGANISM: Leishmania major <220> FEATURE:<221> NAME/KEY: VARIANT <222> LOCATION: (1)...(146)<223> OTHER INFORMATION: Xaa = Any Amino Aci #d <400> SEQUENCE: 36Gly Thr Ser Pro Cys Leu His Leu Leu Ala As #p Ile Arg Gly Glu Phe 1               5   #                10   #                15Phe Asn Leu Arg Arg Val Glu Leu Leu Asn Va #l Ala Gln Gln Ala Gln            20       #            25       #            30Leu Val Val Ala His Glu Gly Asp Gly Gln Se #r Ser Ala Ala His Ala        35           #        40           #        45Ala Ser Ala Ala Asp Ala Val Asn Val Leu Le #u Thr Arg Glu Arg Gln    50               #    55               #    60Ile Val Ala Glu Asp Glu Arg Asp Ala Leu Hi #s Ile Asn Ala Thr Arg65                   #70                   #75                   #80Pro Gln Val Arg Cys Asn Xaa His Ala Ala Va #l Ser Ile Thr Glu Cys                85   #                90   #                95Arg Ile Ile Ser Ser Arg Ser Ala Trp Gly Me #t Ser Pro Cys Met Ala            100       #           105       #           110Asp Thr Ala Lys Phe Ser Arg Val Ile Phe Le #u Ala Ser Cys Ser Thr        115           #       120           #       125Phe Leu Arg Val Leu Xaa Lys Thr Thr Ala Tr #p Ala Ile Val Lys Leu    130               #   135               #   140 Ser Tyr 145<210> SEQ ID NO 37 <211> LENGTH: 77 <212> TYPE: PRT<213> ORGANISM: Leishmania major <220> FEATURE: <221> NAME/KEY: VARIANT<222> LOCATION: (1)...(77) <223> OTHER INFORMATION: Xaa = Any Amino Aci#d <400> SEQUENCE: 37 Ala Gln Gly Phe Arg Val Ile Phe Thr His Gl#y Gly Ala Gln Met Gly  1               5   #                10  #                15 Glu Val Asn Thr Arg Thr Glu Leu Leu Asp Hi#s Ile Asn Gln Ile Val             20       #            25      #            30 Val Ser Thr His Ala Val Xaa Thr Gly Ala Hi#s Gly Lys Thr Val Cys         35           #        40          #        45 His Ala Val Tyr Gly Ile Asn His Pro Leu Hi#s Ile Phe Asn Gly Gly     50               #    55              #    60 Asn Asn Ala Arg Gln Ala Glu Asp Pro Ala Ar #g Leu Ile65                   #70                   #75 <210> SEQ ID NO 38<211> LENGTH: 68 <212> TYPE: PRT <213> ORGANISM: Leishmania major<220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: (1)...(68)<223> OTHER INFORMATION: Xaa = Any Amino Aci #d <400> SEQUENCE: 38His Glu Pro Gln Trp Ser Ser Met Lys Ile Le #u Gln Tyr Leu Thr Leu 1               5   #                10   #                15Asp Gly Thr Gln Val Ser Gly Thr Leu Pro Pr #o Gln Trp Ser Ala Met            20       #            25       #            30Ala Ser Val Arg Ile Leu Asn Leu Xaa Gly Th #r Glu Val Ser Gly Thr        35           #        40           #        45Leu Pro Pro Glu Trp Ile Ser Met Xaa Arg Le #u Gln Thr Leu Asn Leu    50               #    55               #    60 Arg Arg Thr Lys 65<210> SEQ ID NO 39 <211> LENGTH: 65 <212> TYPE: PRT<213> ORGANISM: Leishmania major <220> FEATURE: <221> NAME/KEY: VARIANT<222> LOCATION: (1)...(65) <223> OTHER INFORMATION: Xaa = Any Amino Aci#d <400> SEQUENCE: 39 Ala Arg Glu Ala Gln Leu Ala His Arg Ile Cy#s Asp Tyr Leu Pro Glu  1               5   #                10  #                15 Gln Gly Gln Xaa Phe Val Gly Asn Ser Leu Va#l Val Arg Leu Ile Asp             20       #            25      #            30 Xaa Leu Xaa Gln Xaa Pro Ala Gly Tyr Pro Va#l Tyr Xaa Asn Arg Gly         35           #        40          #        45 Ala Xaa Gly Ile Xaa Xaa Leu Leu Ser Thr Al#a Ala Gly Val Xaa Arg     50               #    55              #    60 Ala 65 <210> SEQ ID NO 40 <211> LENGTH: 78 <212> TYPE: PRT<213> ORGANISM: Leishmania major <220> FEATURE: <221> NAME/KEY: VARIANT<222> LOCATION: (1)...(78) <223> OTHER INFORMATION: Xaa = Any Amino Aci#d <400> SEQUENCE: 40 Asp Asp His Ala Pro Lys Glu Asp Gly His Al#a Pro Lys Asn Asp Asp  1               5   #                10  #                15 His Ala Pro Lys Glu Asp Gly His Ala Pro Ly#s Asn Asp Asp His Ala             20       #            25      #            30 Pro Lys Glu Asp Gly His Ala Pro Lys Asn As#p Gly Asp Val Gln Xaa         35           #        40          #        45 Lys Ser Glu Asp Gly Asp Asn Val Gly Glu Gl#y Gly Lys Gly Asn Glu     50               #    55              #    60 Asp Gly Asn Asp Asp Gln Pro Lys Glu His Al #a Ala Gly Asn65                   #70                   #75 <210> SEQ ID NO 41<211> LENGTH: 169 <212> TYPE: PRT <213> ORGANISM: Leishmania major<400> SEQUENCE: 41 Leu Gln Gln Arg Leu Asp Thr Ala Thr Gln Gl#n Arg Ala Glu Leu Glu  1               5   #                10  #                15 Ala Arg Val Ala Arg Leu Ala Ala Asp Arg As#p Glu Ala Arg Gln Gln             20       #            25      #            30 Leu Ala Ala Asn Ala Glu Glu Leu Gln Gln Ar#g Leu Asp Thr Ala Thr         35           #        40          #        45 Gln Gln Arg Ala Glu Leu Glu Ala Arg Val Al#a Arg Leu Ala Ala Asp     50               #    55              #    60 Gly Asp Glu Ala Arg Gln Gln Leu Ala Ala As#n Ala Glu Glu Leu Gln 65                   #70                  #75                   #80 Gln Arg Leu Asp Thr Ala Thr Gln Gln Arg Al#a Glu Leu Glu Ala Gln                 85   #                90  #                95 Val Ala Arg Leu Ala Ala Asn Ala Glu Glu Le#u Gln Gln Arg Leu Asp             100       #           105      #           110 Thr Ala Thr Gln Gln Arg Ala Glu Leu Glu Al#a Arg Val Ala Arg Leu         115           #       120          #       125 Ala Ala Asp Arg Asp Glu Ala Arg Gln Gln Le#u Ala Ala Asn Ala Glu     130               #   135              #   140 Glu Leu Gln Gln Arg Leu Asp Thr Ala Thr Gl#n Gln Arg Ala Glu Leu 145                 1 #50                 1#55                 1 #60 Glu Ala Gln Val Ala Arg Leu Ala Ala                165 <210> SEQ ID NO 42 <211> LENGTH: 98 <212> TYPE: PRT<213> ORGANISM: Leishmania major <220> FEATURE: <221> NAME/KEY: VARIANT<222> LOCATION: (1)...(98) <223> OTHER INFORMATION: Xaa = Any Amino Aci#d <400> SEQUENCE: 42 Ala Arg Xaa Ile Phe Val Lys Thr Leu Thr Gl#y Xaa Thr Ile Ala Leu  1               5   #                10  #                15 Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Va#l Lys Ala Lys Ile Gln             20       #            25      #            30 Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Ar#g Leu Ile Phe Ala Gly         35           #        40          #        45 Lys Gln Leu Glu Xaa Gly Arg Thr Leu Ser As#p Tyr Asn Ile Gln Lys     50               #    55              #    60 Glu Ser Thr Leu His Leu Val Leu Arg Leu Ar#g Gly Gly Met Xaa Ile 65                   #70                  #75                   #80 Phe Val Lys Thr Leu Thr Gly Xaa Thr Ile Al#a Leu Glu Val Glu Pro                 85   #                90  #                95 Asn Asp <210> SEQ ID NO 43 <211> LENGTH: 39<212> TYPE: PRT <213> ORGANISM: Leishmania major <400> SEQUENCE: 43Leu Gln Gln Arg Leu Asp Thr Ala Thr Gln Gl #n Arg Ala Glu Leu Glu 1               5   #                10   #                15Ala Arg Val Ala Arg Leu Ala Ala Asp Arg As #p Glu Ala Arg Gln Gln            20       #            25       #            30Leu Ala Ala Asn Ala Glu Glu         35 <210> SEQ ID NO 44<211> LENGTH: 600 <212> TYPE: DNA <213> ORGANISM: Leishmania chagasi<220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)...(600)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 44cggccgcctc agcgaggagg agatcgagcg catggtgcgc gaggctgccg ag#ttcgagga     60tgaggaccgc aaggtgcgcg aacgtgtcga agcgaagaac tcgctagaga gc#atcgcgta    120ctcgcttcgc aaccagatca acgacaagga caagcttggt gacaagctcg cc#gcggacga    180caagaaggcg atcgaggagg ctgtgaagga tgccctcgac tttgtccacg ag#aaccccaa    240tgcagaccgt gaggagttcg aggctgctcg cacgaagctg cagagtgtga cg#aaccccat    300cattcaaaag gtgtaccagg gcgccgccgg ctctggtgca gaagaggcgg ac#gcgatgga    360tgacttgtta gtcggccgcg tgaaaagaaa aacagggaaa gcgggaacat nc#cacaanaa    420ccnaagaaga aagggggtng cgacaccgct cgaacaccga cggcncacat nc#ntcatggg    480catgctcagc tttcctctcc ccaacaaacc agaaggtttt ctccaaacnc cg#tctcngcn    540cccaaaatac ggaaangtta ancgaaaaan ccccttccac caattgnngt tc#ttttgttt    600 <210> SEQ ID NO 45 <211> LENGTH: 1748 <212> TYPE: DNA<213> ORGANISM: Leishmania chagasi <400> SEQUENCE: 45ctagtggatc ccccgggctg caggaattca cggaatacgt acctcctccc cc#ttcttggt     60agaagaacaa caacaacgtt caagacgacg ccgcgccttc ttgtaccgca tt#tgcttctg    120agcacgttca atccgtgcct tgcaaacatg gaggcgtaca agaagctgga aa#cgatcttt    180acgaaggtct accgcctgga ccacttcctc ggtctgggca actgggacat ga#acacaaac    240atgcccccca agggcgagga atcacgcggt gaggcgatgg cgatgctctc gg#agctccgc    300tttggcttca tcacggcacc ggaggtgaaa agcctgattg agagtgccac ca#agggcagc    360gaggagctga atgcggtgca gcgcgctaac ttgcgggaga tgaggcgtgc gt#ggaagagc    420gccaccgcct tgccggctga gtttgtgggc cgcaagatgc gcctcacgac ac#acgcgcac    480agcgtgtggc gcgacagccg caaagcaaat gacttcgcca agttcctacc gg#tgctcagg    540gacctggtgg cgctcgcccg tgaggagggc tcatacctcg ccgccggcac ct#ccctctcc    600ccgtatgagg cgctcatgaa cgagtacgag ccaggaatca cgacacaaaa gc#tggatgag    660gtgtacgcaa atgtaaagtc gtggctgccg cagctgctaa aggacattgt gc#agaagcag    720tccggcgagt cggtgattgc gttctcgcat aagttcccgc aggacaagca gg#aagcactg    780tgcaaggaat tcatgaagat ctggcacttc gacaccgatg ccggtcgcct cg#acgtcagc    840ccccaccctt tcacgggaat gacgaaggag gactgccgac tcacaacaaa ct#acatcgaa    900gacacgtttg ttcagagctt gtatggcgtc atccacgaga gtgggcatgg ca#agtacgag    960cagaactgtg gcccacgcga gcacatcacg cagccggtgt gcaacgcccg ct#ctcttggc   1020ctgcatgaga gccagagcct ctttgcggag tttcagatcg gccacgcgac gc#ccttcatc   1080gactacctca caactcgcct tcctgagttc ttcgaggcgc agccagcgtt ct#cgcaggac   1140aacatgcgca agtcgctgca gcaggtgaag ccgggctaca ttcgcgtcga tg#ccgatgag   1200gtgtgctacc ctctgcacgt gatcctgcgc tacgagatcg agcgcgactt ga#tggagggc   1260aaaatggagg tggaagacgt gccgcgcgcg tggaacgcaa agatgcagga gt#acttgggt   1320ctctcaacgg agggccgtga cgacgttggg tgcctgcagg acgtgcattg gt#ccatggtg   1380cgctcggcta ctctccgacg tactcgctcg gcgccatgta tgcggcgcag at#catggcga   1440gcatccgaaa ggagctggga gacgacaagg tggatgagtg cctgcgcacc gg#tgagctcg   1500gccccctcct ggaaaagcag caggagaaga tctgggatca tgggtgcctg ta#cgagacgg   1560acgacctcat gacgcgtgcg acgggcgaga cgctgaaccc cgagtacctg cg#ccgccacc   1620tggaggcgcg ctacataaac gcctgagtcg cgagcggttg acacacgcgc tc#gctagcac   1680atgacgcgtc tttattattc tttgttgtgc attcggaatt ccgcggaatt cg#atatcaag   1740 cttatcga                 #                  #                   #        1748 <210> SEQ ID NO 46 <211> LENGTH: 560<212> TYPE: DNA <213> ORGANISM: Leishmania chagasi <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(560)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 46cggaaggagg atggccatac acagaaaaat gacggcgatg gccctaagga gg#acggccgt     60acacagaaaa acgacgacgg tggccctaag gaggacggcc atacacagaa aa#atgacggc    120gatggcccta aggaggacgg ccgtacacag aaaaataacg gcgatggccc tn#aggaggac    180ggccatacac agaaaaatga cggcgatgcc cctnaggagg acggccgtac ac#anaaaaat    240gacggcnatg gccctnagga ggacggccgt acacagaaaa atgacngcca tg#gcccttag    300gangacgccg tacacagaaa aatgacgcna tggccctnag ggaggacggc ca#tacccana    360aaaattgacg gcnatngccc ttaggangac ggccgtnccc anaaanantg ac#ngcggtng    420cccttaagga agatgaaaat ctgccaccaa aacnattggg aatgcncagg aa#aanaacna    480anatngaccc cacgtggggg atgganctta cngcnattaa nattgttacc at#tatcnacc    540 naaggacnng ttgccgncaa             #                  #                   #560 <210> SEQ ID NO 47 <211> LENGTH: 600<212> TYPE: DNA <213> ORGANISM: Leishmania chagasi <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(600)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 47cgtccgagaa acccgtacat gtatgctgct ggtagaaggc gcagagctgg tc#cctctgat     60gcacaagcat gaggtcgtac attgcctggt tcgtcatttt ccagagcaca ac#gagcagcg    120tcatcataca gcatccaata gccgccagag tgaatgcgat gcgcacacca ag#tcgaaagt    180ggtcgaccag taggggaatg tgaccctggc tggcgtgcaa catgatcgcc ac#gccagcgg    240tgggccacac cacaacagag gcgacgaaag agaacatgaa cttgctcacg aa#gctnacaa    300taagggcgtc gctngtgatg ctaagaacca cgccnaggta gacggcgaag an#caaactaa    360acacaagcgt gacgatcccg aaaagaagga tctctgcgga attttcgtga ga#taganaat    420gcccgtactg gaaaaanaag ccggcaggcg cgcgataacg ctgcaacttg cc#gctcctcg    480cgggcgcgtt ttcgctcctt ctccgacttg atggcgcngt cngncttgac aa#aacggtta    540agctcctcat gccccagccg attcccagct cacggtccac ttccggccat gc#ccacggac    600 <210> SEQ ID NO 48 <211> LENGTH: 1053 <212> TYPE: DNA<213> ORGANISM: Leishmania chagasi <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(1053)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 48gggaaaaaag tggagctcca ccgcggtggc ggccgctcta gaactagtgg at#cccccggg     60ctgcaggaat tccgcggaat tccgcggaat tccgcggaat tccgtccgac gc#ggcacccg    120cacaggggtc gacagtgacg caacctcctc caccactgcg gcctacgacg gc#gccggctc    180cgcgccagtg atggttgacg ccaatgtgag ccaccctccg tacgcggggc at#gaccaagt    240gtacatgcac gtcggcaagc ccatcgtggg caacaccctc gacggataca ac#gggtgcgt    300gttcgcctac gggcanacgg gcagcggcaa aaccttcacg atgctcggnt ac#gcgccgag    360cacgancgac atccgcgctc gcaaagggtc cgtcccctgc ggggccagca gc#atggagaa    420cagcactcct cttgacagcg ctgtggagcc gtttgagagc gatgacggcg ac#gacgtggt    480ggacaagacg gggctggatc cgaacgagct gcaaggcatc atcccgcgcg cg#tgcacgga    540cctgttcgat ggtctccgtg cgaagcgcgc caaggactcc gacttcacgt ac#cgcgtgga    600ggtgtcttac tacgagatct acaacgagaa ggtgttcgat ctcatccggc cg#cagcgcaa    660cacggacctg aggatacgta actcgcccaa ctccggtcca tttatcgaag gc#ctgacgtg    720gaagatggtg tccaaggagg aagacgtcgc ccgcgtgatt cgcaagggca tg#caggagcg    780ccacacggct gcgaccaagt tcaacgaccg cagcagccgc agccacgcca tc#ctcacctt    840caacattgtg cagctgtcga tggacgactc cgacaacgcg ttccagatgc gc#agcaagct    900gaacctggtg gaccttgctg ggtcggagcg cactggtgcg gccggagccg ag#ggcaatga    960gttccacgac ggtgtgaaga tcaaccactc gctgacggtg ctggggcgcg tg#atcgaccg   1020 tctggcggac ctctcgcaga acaagggagg ggg       #                   #       1053 <210> SEQ ID NO 49 <211> LENGTH: 136<212> TYPE: PRT <213> ORGANISM: Leishmania chagasi <400> SEQUENCE: 49Gly Arg Leu Ser Glu Glu Glu Ile Glu Arg Me #t Val Arg Glu Ala Ala 1               5   #                10   #                15Glu Phe Glu Asp Glu Asp Arg Lys Val Arg Gl #u Arg Val Glu Ala Lys            20       #            25       #            30Asn Ser Leu Glu Ser Ile Ala Tyr Ser Leu Ar #g Asn Gln Ile Asn Asp        35           #        40           #        45Lys Asp Lys Leu Gly Asp Lys Leu Ala Ala As #p Asp Lys Lys Ala Ile    50               #    55               #    60Glu Glu Ala Val Lys Asp Ala Leu Asp Phe Va #l His Glu Asn Pro Asn65                   #70                   #75                   #80Ala Asp Arg Glu Glu Phe Glu Ala Ala Arg Th #r Lys Leu Gln Ser Val                85   #                90   #                95Thr Asn Pro Ile Ile Gln Lys Val Tyr Gln Gl #y Ala Ala Gly Ser Gly            100       #           105       #           110Ala Glu Glu Ala Asp Ala Met Asp Asp Leu Le #u Val Gly Arg Val Lys        115           #       120           #       125Arg Lys Thr Gly Lys Ala Gly Thr     130               #   135<210> SEQ ID NO 50 <211> LENGTH: 510 <212> TYPE: PRT<213> ORGANISM: Leishmania chagasi <400> SEQUENCE: 50Tyr Leu Leu Pro Leu Leu Gly Arg Arg Thr Th #r Thr Thr Phe Lys Thr 1               5   #                10   #                15Thr Pro Arg Leu Leu Val Pro His Leu Leu Le #u Ser Thr Phe Asn Pro            20       #            25       #            30Cys Leu Ala Asn Met Glu Ala Tyr Lys Lys Le #u Glu Thr Ile Phe Thr        35           #        40           #        45Lys Val Tyr Arg Leu Asp His Phe Leu Gly Le #u Gly Asn Trp Asp Met    50               #    55               #    60Asn Thr Asn Met Pro Pro Lys Gly Glu Glu Se #r Arg Gly Glu Ala Met65                   #70                   #75                   #80Ala Met Leu Ser Glu Leu Arg Phe Gly Phe Il #e Thr Ala Pro Glu Val                85   #                90   #                95Lys Ser Leu Ile Glu Ser Ala Thr Lys Gly Se #r Glu Glu Leu Asn Ala            100       #           105       #           110Val Gln Arg Ala Asn Leu Arg Glu Met Arg Ar #g Ala Trp Lys Ser Ala        115           #       120           #       125Thr Ala Leu Pro Ala Glu Phe Val Gly Arg Ly #s Met Arg Leu Thr Thr    130               #   135               #   140His Ala His Ser Val Trp Arg Asp Ser Arg Ly #s Ala Asn Asp Phe Ala145                 1 #50                 1 #55                 1 #60Lys Phe Leu Pro Val Leu Arg Asp Leu Val Al #a Leu Ala Arg Glu Glu                165   #               170   #               175Gly Ser Tyr Leu Ala Ala Gly Thr Ser Leu Se #r Pro Tyr Glu Ala Leu            180       #           185       #           190Met Asn Glu Tyr Glu Pro Gly Ile Thr Thr Gl #n Lys Leu Asp Glu Val        195           #       200           #       205Tyr Ala Asn Val Lys Ser Trp Leu Pro Gln Le #u Leu Lys Asp Ile Val    210               #   215               #   220Gln Lys Gln Ser Gly Glu Ser Val Ile Ala Ph #e Ser His Lys Phe Pro225                 2 #30                 2 #35                 2 #40Gln Asp Lys Gln Glu Ala Leu Cys Lys Glu Ph #e Met Lys Ile Trp His                245   #               250   #               255Phe Asp Thr Asp Ala Gly Arg Leu Asp Val Se #r Pro His Pro Phe Thr            260       #           265       #           270Gly Met Thr Lys Glu Asp Cys Arg Leu Thr Th #r Asn Tyr Ile Glu Asp        275           #       280           #       285Thr Phe Val Gln Ser Leu Tyr Gly Val Ile Hi #s Glu Ser Gly His Gly    290               #   295               #   300Lys Tyr Glu Gln Asn Cys Gly Pro Arg Glu Hi #s Ile Thr Gln Pro Val305                 3 #10                 3 #15                 3 #20Cys Asn Ala Arg Ser Leu Gly Leu His Glu Se #r Gln Ser Leu Phe Ala                325   #               330   #               335Glu Phe Gln Ile Gly His Ala Thr Pro Phe Il #e Asp Tyr Leu Thr Thr            340       #           345       #           350Arg Leu Pro Glu Phe Phe Glu Ala Gln Pro Al #a Phe Ser Gln Asp Asn        355           #       360           #       365Met Arg Lys Ser Leu Gln Gln Val Lys Pro Gl #y Tyr Ile Arg Val Asp    370               #   375               #   380Ala Asp Glu Val Cys Tyr Pro Leu His Val Il #e Leu Arg Tyr Glu Ile385                 3 #90                 3 #95                 4 #00Glu Arg Asp Leu Met Glu Gly Lys Met Glu Va #l Glu Asp Val Pro Arg                405   #               410   #               415Ala Trp Asn Ala Lys Met Gln Glu Tyr Leu Gl #y Leu Ser Thr Glu Gly            420       #           425       #           430Arg Asp Asp Val Gly Cys Leu Gln Asp Val Hi #s Trp Ser Met Val Arg        435           #       440           #       445Ser Ala Thr Leu Arg Arg Thr Arg Ser Ala Pr #o Cys Met Arg Arg Arg    450               #   455               #   460Ser Trp Arg Ala Ser Glu Arg Ser Trp Glu Th #r Thr Arg Trp Met Ser465                 4 #70                 4 #75                 4 #80Ala Cys Ala Pro Val Ser Ser Ala Pro Ser Tr #p Lys Ser Ser Arg Arg                485   #               490   #               495Arg Ser Gly Ile Met Gly Ala Cys Thr Arg Ar #g Thr Thr Ser            500       #           505       #           510<210> SEQ ID NO 51 <211> LENGTH: 107 <212> TYPE: PRT<213> ORGANISM: Leishmania chagasi <220> FEATURE:<221> NAME/KEY: VARIANT <222> LOCATION: (1)...(107)<223> OTHER INFORMATION: Xaa = Any Amino Aci #d <400> SEQUENCE: 51Gly Arg Arg Met Ala Ile His Arg Lys Met Th #r Ala Met Ala Leu Arg 1               5   #                10   #                15Arg Thr Ala Val His Arg Lys Thr Thr Thr Va #l Ala Leu Arg Arg Thr            20       #            25       #            30Ala Ile His Arg Lys Met Thr Ala Met Ala Le #u Arg Arg Thr Ala Val        35           #        40           #        45His Arg Lys Ile Thr Ala Met Ala Leu Arg Ar #g Thr Ala Ile His Arg    50               #    55               #    60Lys Met Thr Ala Met Pro Leu Arg Arg Thr Al #a Val His Xaa Lys Met65                   #70                   #75                   #80Thr Ala Met Ala Leu Arg Arg Thr Ala Val Hi #s Arg Lys Met Thr Ala                85   #                90   #                95Met Ala Leu Arg Xaa Thr Pro Tyr Thr Glu Ly #s             100      #           105 <210> SEQ ID NO 52 <211> LENGTH: 63 <212> TYPE: PRT<213> ORGANISM: Leishmania chagasi <400> SEQUENCE: 52Val Arg Glu Thr Arg Thr Cys Met Leu Leu Va #l Glu Gly Ala Glu Leu 1               5   #                10   #                15Val Pro Leu Met His Lys His Glu Val Val Hi #s Cys Leu Val Arg His            20       #            25       #            30Phe Pro Glu His Asn Glu Gln Arg His His Th #r Ala Ser Asn Ser Arg        35           #        40           #        45Gln Ser Glu Cys Asp Ala His Thr Lys Ser Ly #s Val Val Asp Gln    50               #    55               #    60 <210> SEQ ID NO 53<211> LENGTH: 324 <212> TYPE: PRT <213> ORGANISM: Leishmania chagasi<220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: (1)...(324)<223> OTHER INFORMATION: Xaa = Any Amino Aci #d <400> SEQUENCE: 53Phe Arg Gly Ile Pro Arg Asn Ser Val Arg Ar #g Gly Thr Arg Thr Gly 1               5   #                10   #                15Val Asp Ser Asp Ala Thr Ser Ser Thr Thr Al #a Ala Tyr Asp Gly Ala            20       #            25       #            30Gly Ser Ala Pro Val Met Val Asp Ala Asn Va #l Ser His Pro Pro Tyr        35           #        40           #        45Ala Gly His Asp Gln Val Tyr Met His Val Gl #y Lys Pro Ile Val Gly    50               #    55               #    60Asn Thr Leu Asp Gly Tyr Asn Gly Cys Val Ph #e Ala Tyr Gly Xaa Thr65                   #70                   #75                   #80Gly Ser Gly Lys Thr Phe Thr Met Leu Gly Ty #r Ala Pro Ser Thr Xaa                85   #                90   #                95Asp Ile Arg Ala Arg Lys Gly Ser Val Pro Cy #s Gly Ala Ser Ser Met            100       #           105       #           110Glu Asn Ser Thr Pro Leu Asp Ser Ala Val Gl #u Pro Phe Glu Ser Asp        115           #       120           #       125Asp Gly Asp Asp Val Val Asp Lys Thr Gly Le #u Asp Pro Asn Glu Leu    130               #   135               #   140Gln Gly Ile Ile Pro Arg Ala Cys Thr Asp Le #u Phe Asp Gly Leu Arg145                 1 #50                 1 #55                 1 #60Ala Lys Arg Ala Lys Asp Ser Asp Phe Thr Ty #r Arg Val Glu Val Ser                165   #               170   #               175Tyr Tyr Glu Ile Tyr Asn Glu Lys Val Phe As #p Leu Ile Arg Pro Gln            180       #           185       #           190Arg Asn Thr Asp Leu Arg Ile Arg Asn Ser Pr #o Asn Ser Gly Pro Phe        195           #       200           #       205Ile Glu Gly Leu Thr Trp Lys Met Val Ser Ly #s Glu Glu Asp Val Ala    210               #   215               #   220Arg Val Ile Arg Lys Gly Met Gln Glu Arg Hi #s Thr Ala Ala Thr Lys225                 2 #30                 2 #35                 2 #40Phe Asn Asp Arg Ser Ser Arg Ser His Ala Il #e Leu Thr Phe Asn Ile                245   #               250   #               255Val Gln Leu Ser Met Asp Asp Ser Asp Asn Al #a Phe Gln Met Arg Ser            260       #           265       #           270Lys Leu Asn Leu Val Asp Leu Ala Gly Ser Gl #u Arg Thr Gly Ala Ala        275           #       280           #       285Gly Ala Glu Gly Asn Glu Phe His Asp Gly Va #l Lys Ile Asn His Ser    290               #   295               #   300Leu Thr Val Leu Gly Arg Val Ile Asp Arg Le #u Ala Asp Leu Ser Gln305                 3 #10                 3 #15                 3 #20Asn Lys Gly Gly <210> SEQ ID NO 54 <211> LENGTH: 1585 <212> TYPE: DNA<213> ORGANISM: Leishmania major <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(1585)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 54aaagctggag ctccaccgcg gtggcggccg ctctagaact agtggatccc cc#gggctgca     60ggaattcggc acgagtgctg cccgacatga catgctcgct gaccggactt ca#gtgcacag    120acccgaactg caagacctgc acaacttacg gtcagtgcac agactgcaac ga#cggctacg    180gtctcacctc ctccagcgtt tgcgtgcgct gcagtgtagc gggctgcaag ag#ctgccccg    240tcgacgctaa cgtctgcaaa gtgtgtctcg gcggcagcga gccgatcaac aa#tatgtgcc    300cctgcaccga ccccaactgc gccagctgcc ccagcgacgc tggcacgtgc ac#tcagtgcg    360cgaacggcta cggtctcgtg gacggcgcct gtgtgagatg ccaggagccc aa#ctgcttca    420gctgcgacag cgacgcgaat aagtgcacac aatgtgcgcc gaactactac ct#caccccgc    480tcttgacctg ctccccggtg gcctgcaaca tcgagcactg catgcagtgc ga#cccacaga    540cgccgtcgcg ctgccaggag tgcgtgtccc cctacgtggt tgacagctac ga#cggcctct    600gcaggctctc cgatgcctgc tccgtgccca actgcaagaa gtgcgagacc gg#tacctcca    660ggctctgcgc cgagtgcgac accggctaca gtctctccgc cgacgcgacg ag#ctgcagca    720gtccaaccac gcagccgtgc gaggtggagc actgcaacac atgtgtgaac gg#cgatagca    780cccgctgtgc ctactgcaac accggctact acgtctccga tggcaagtgc aa#ggccatgc    840agggctgcta cgtgtcgaac tgcgcgcagt gcatgctgct tgacagcacc aa#gtgctcca    900cgtgcgtgaa agggtacctg ctcacgtcgt cctacagttg cgtctcgcag aa#agtcatca    960acagtgcggc cgcgccctac tctctgtggg tggccgccgc cgtgctcctc ac#ctcttttg   1020ccatgcacct agcatagtgc gcagcggcat gcgaacaacc ccactctcat tc#tccaacat   1080gtgcatacac acacacacag acagcggggc agcaccccct ccccacacac ac#acacgcac   1140ttcccccttg tcttgttctt ctttcctcgn ttcgcatttc tttctctcgt gc#gctggcgc   1200cggcctcctg cacgtcgctc ccctccccct aacctctatt ctctctctct ct#ctctctcg   1260ccggcatcat tgcttcttac ccttttctga tccttgctcg cgtgggcgga ca#ctgccaca   1320gtcccacagc gcagacacac gtgtttaaac ggcgcaggca tccctcccta tc#acttcatt   1380tctcctaaag ccactcacca agtcgcacac cgccctcccc catcggccgc cc#ttccgggc   1440gcagctgtgc ggaatgggtg tgtgctcgac ctcgttcctg gcagctcact cg#catgtgta   1500cagccactcc aaccacgaaa gctctcttct gcgcacataa aaaaaaaaaa aa#aaaaaaaa   1560 ctcgaggggg ggcccggtac ccaaa          #                   #             1585 <210> SEQ ID NO 55<211> LENGTH: 320 <212> TYPE: PRT <213> ORGANISM: Leishmania major<400> SEQUENCE: 55 Val Leu Pro Asp Met Thr Cys Ser Leu Thr Gl#y Leu Gln Cys Thr Asp  1               5   #                10  #                15 Pro Asn Cys Lys Thr Cys Thr Thr Tyr Gly Gl#n Cys Thr Asp Cys Asn             20       #            25      #            30 Asp Gly Tyr Gly Leu Thr Ser Ser Ser Val Cy#s Val Arg Cys Ser Val         35           #        40          #        45 Ala Gly Cys Lys Ser Cys Pro Val Asp Ala As#n Val Cys Lys Val Cys     50               #    55              #    60 Leu Gly Gly Ser Glu Pro Ile Asn Asn Met Cy#s Pro Cys Thr Asp Pro 65                   #70                  #75                   #80 Asn Cys Ala Ser Cys Pro Ser Asp Ala Gly Th#r Cys Thr Gln Cys Ala                 85   #                90  #                95 Asn Gly Tyr Gly Leu Val Asp Gly Ala Cys Va#l Arg Cys Gln Glu Pro             100       #           105      #           110 Asn Cys Phe Ser Cys Asp Ser Asp Ala Asn Ly#s Cys Thr Gln Cys Ala         115           #       120          #       125 Pro Asn Tyr Tyr Leu Thr Pro Leu Leu Thr Cy#s Ser Pro Val Ala Cys     130               #   135              #   140 Asn Ile Glu His Cys Met Gln Cys Asp Pro Gl#n Thr Pro Ser Arg Cys 145                 1 #50                 1#55                 1 #60 Gln Glu Cys Val Ser Pro Tyr Val Val Asp Se#r Tyr Asp Gly Leu Cys                 165   #               170  #               175 Arg Leu Ser Asp Ala Cys Ser Val Pro Asn Cy#s Lys Lys Cys Glu Thr             180       #           185      #           190 Gly Thr Ser Arg Leu Cys Ala Glu Cys Asp Th#r Gly Tyr Ser Leu Ser         195           #       200          #       205 Ala Asp Ala Thr Ser Cys Ser Ser Pro Thr Th#r Gln Pro Cys Glu Val     210               #   215              #   220 Glu His Cys Asn Thr Cys Val Asn Gly Asp Se#r Thr Arg Cys Ala Tyr 225                 2 #30                 2#35                 2 #40 Cys Asn Thr Gly Tyr Tyr Val Ser Asp Gly Ly#s Cys Lys Ala Met Gln                 245   #               250  #               255 Gly Cys Tyr Val Ser Asn Cys Ala Gln Cys Me#t Leu Leu Asp Ser Thr             260       #           265      #           270 Lys Cys Ser Thr Cys Val Lys Gly Tyr Leu Le#u Thr Ser Ser Tyr Ser         275           #       280          #       285 Cys Val Ser Gln Lys Val Ile Asn Ser Ala Al#a Ala Pro Tyr Ser Leu     290               #   295              #   300 Trp Val Ala Ala Ala Val Leu Leu Thr Ser Ph#e Ala Met His Leu Ala 305                 3 #10                 3#15                 3 #20 <210> SEQ ID NO 56 <211> LENGTH: 14<212> TYPE: PRT <213> ORGANISM: Leishmania major <400> SEQUENCE: 56Pro Lys Glu Asp Gly His Ala Pro Lys Asn As #p Asp His Ala 1               5   #                10 <210> SEQ ID NO 57<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Leishmania major<400> SEQUENCE: 57 Pro Lys Glu Asp Gly His Ala  1               5<210> SEQ ID NO 58 <211> LENGTH: 7 <212> TYPE: PRT<213> ORGANISM: Leishmania major <400> SEQUENCE: 58Pro Lys Asn Asp Asp His Ala  1               5 <210> SEQ ID NO 59<211> LENGTH: 264 <212> TYPE: DNA <213> ORGANISM: Leishmania chagasi<400> SEQUENCE: 59atgcaccatc atcaccatca catgggaagc tcctgcacga aggactccgc aa#aggagccc     60cagaagcgtg ctgataacat cgatacgacc actcgaagcg atgagaagga cg#gcatccat    120gtccaggaga gcgccggtcc tgtgcaggag aacttcgggg atgcgcagga ga#agaacgaa    180gatggacaca acgtggggga tggagctaac gacaatgagg atggtaacga tg#atcagccg    240 aaggagcagg ttgccggcaa ctag          #                   #               264 <210> SEQ ID NO 60<211> LENGTH: 744 <212> TYPE: DNA <213> ORGANISM: Leishmania chagasi<400> SEQUENCE: 60atgggagcct actgcacgaa ggactccgca aaggagcccc agaagcgtgc tg#ataacatc     60cataaaacca ctgaggccaa tcacagaggc gccgccggtg tgcccccgaa gc#acgccggc    120ggtgcgatga acgactctgc cccgaaggag gatggccata cacagaaaaa tg#acggcgat    180ggccctaagg aggacggccg tacacagaaa aacgacgacg gtggccctaa gg#aggacggc    240catacacaga aaaatgacgg cgatggccct aaggaggacg gccgtacaca ga#aaaataac    300ggcgatggcc ctaaggagga cggccataca cagaaaaatg acggcgatgc cc#ctaaggag    360gacggccgta cacagaaaaa tgacggcgat ggccctaagg aggacggccg ta#cacagaaa    420aatgacggcg atggccctaa ggaggacggc cgtacacaga aaaatgacgg cg#atggccct    480aaggaggacg gccgtacaca gaaaaatgac ggcgatggcc ctaaggagga cg#gccataca    540cagaaaaatg acggcgatgg ccctaaggag gacggccgta cacagaaaaa tg#acggcggt    600ggccctaagg aggatgagaa tctgcagcaa aacgatggga atgcgcagga ga#agaacgaa    660gatggacaca acgtggggga tggagctaac ggcaatgagg atggtaacga tg#atcagccg    720 aaggagcagg ttgccggcaa ctag          #                   #               744 <210> SEQ ID NO 61<211> LENGTH: 80 <212> TYPE: PRT <213> ORGANISM: Leishmania chagasi<400> SEQUENCE: 61 Met Gly Ser Ser Cys Thr Lys Asp Ser Ala Ly#s Glu Pro Gln Lys Arg  1               5   #                10  #                15 Ala Asp Asn Ile Asp Thr Thr Thr Arg Ser As#p Glu Lys Asp Gly Ile             20       #            25      #            30 His Val Gln Glu Ser Ala Gly Pro Val Gln Gl#u Asn Phe Gly Asp Ala         35           #        40          #        45 Gln Glu Lys Asn Glu Asp Gly His Asn Val Gl#y Asp Gly Ala Asn Asp     50               #    55              #    60 Asn Glu Asp Gly Asn Asp Asp Gln Pro Lys Gl#u Gln Val Ala Gly Asn 65                   #70                  #75                   #80 <210> SEQ ID NO 62 <211> LENGTH: 247<212> TYPE: PRT <213> ORGANISM: Leishmania chagasi <400> SEQUENCE: 62Met Gly Ala Tyr Cys Thr Lys Asp Ser Ala Ly #s Glu Pro Gln Lys Arg 1               5   #                10   #                15Ala Asp Asn Ile His Lys Thr Thr Glu Ala As #n His Arg Gly Ala Ala            20       #            25       #            30Gly Val Pro Pro Lys His Ala Gly Gly Ala Me #t Asn Asp Ser Ala Pro        35           #        40           #        45Lys Glu Asp Gly His Thr Gln Lys Asn Asp Gl #y Asp Gly Pro Lys Glu    50               #    55               #    60Asp Gly Arg Thr Gln Lys Asn Asp Asp Gly Gl #y Pro Lys Glu Asp Gly65                   #70                   #75                   #80His Thr Gln Lys Asn Asp Gly Asp Gly Pro Ly #s Glu Asp Gly Arg Thr                85   #                90   #                95Gln Lys Asn Asn Gly Asp Gly Pro Lys Glu As #p Gly His Thr Gln Lys            100       #           105       #           110Asn Asp Gly Asp Ala Pro Lys Glu Asp Gly Ar #g Thr Gln Lys Asn Asp        115           #       120           #       125Gly Asp Gly Pro Lys Glu Asp Gly Arg Thr Gl #n Lys Asn Asp Gly Asp    130               #   135               #   140Gly Pro Lys Glu Asp Gly Arg Thr Gln Lys As #n Asp Gly Asp Gly Pro145                 1 #50                 1 #55                 1 #60Lys Glu Asp Gly Arg Thr Gln Lys Asn Asp Gl #y Asp Gly Pro Lys Glu                165   #               170   #               175Asp Gly His Thr Gln Lys Asn Asp Gly Asp Gl #y Pro Lys Glu Asp Gly            180       #           185       #           190Arg Thr Gln Lys Asn Asp Gly Gly Gly Pro Ly #s Glu Asp Glu Asn Leu        195           #       200           #       205Gln Gln Asn Asp Gly Asn Ala Gln Glu Lys As #n Glu Asp Gly His Asn    210               #   215               #   220Val Gly Asp Gly Ala Asn Gly Asn Glu Asp Gl #y Asn Asp Asp Gln Pro225                 2 #30                 2 #35                 2 #40Lys Glu Gln Val Ala Gly Asn                 245 <210> SEQ ID NO 63<211> LENGTH: 14 <212> TYPE: PRT <213> ORGANISM: Leishmania chagasi<220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: (6)...(6)<223> OTHER INFORMATION: Xaa = His or Arg <221> NAME/KEY: VARIANT<222> LOCATION: (12)...(12) <223> OTHER INFORMATION: Xaa = Gly or Asp<221> NAME/KEY: VARIANT <222> LOCATION: (13)...(13)<223> OTHER INFORMATION: Xaa = Asp or Gly <400> SEQUENCE: 63Pro Lys Glu Asp Gly Xaa Thr Gln Lys Asn As #p Xaa Xaa Gly 1               5   #                10 <210> SEQ ID NO 64<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Leishmania chagasi<220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: (6)...(6)<223> OTHER INFORMATION: Xaa = His or Arg <400> SEQUENCE: 64Pro Lys Glu Asp Gly Xaa Thr  1               5 <210> SEQ ID NO 65<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Leishmania chagasi<220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: (5)...(5)<223> OTHER INFORMATION: Xaa = Gly or Asp <221> NAME/KEY: VARIANT<222> LOCATION: (6)...(6) <223> OTHER INFORMATION: Xaa = Asp or Gly<400> SEQUENCE: 65 Gln Lys Asn Asp Xaa Xaa Gly  1               5<210> SEQ ID NO 66 <211> LENGTH: 17 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic peptide to asse#s diagnostic potential       of repeat in Lc Gene B <400> SEQUENCE: 66Gly Cys Gly Pro Lys Glu Asp Gly Arg Thr Gl #n Lys Asn Asp Gly Asp 1               5   #                10   #                15  Gly<210> SEQ ID NO 67 <211> LENGTH: 31 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic peptide to asse#s diagnostic potential       of repeat in Lc Gene B <400> SEQUENCE: 67Gly Cys Gly Pro Lys Glu Asp Gly Arg Thr Gl #n Lys Asn Asp Gly Asp 1               5   #                10   #                15Gly Pro Lys Glu Asp Gly Arg Thr Gln Lys As #n Asp Gly Asp Gly            20       #            25       #            30<210> SEQ ID NO 68 <211> LENGTH: 45 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic peptide to asse#s diagnostic potential       of repeat in Lc Gene B <400> SEQUENCE: 68Gly Cys Gly Pro Lys Glu Asp Gly Arg Thr Gl #n Lys Asn Asp Gly Asp 1               5   #                10   #                15Gly Pro Lys Glu Asp Gly Arg Thr Gln Lys As #n Asp Gly Asp Gly Pro            20       #            25       #            30Lys Glu Asp Gly Arg Thr Gln Lys Asn Asp Gl #y Asp Gly        35           #        40           #        45<210> SEQ ID NO 69 <211> LENGTH: 17 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic peptide to asse#s diagnostic potential       of repeat in Lc Gene B <400> SEQUENCE: 69Gly Cys Gly Pro Lys Glu Asp Gly His Thr Gl #n Lys Asn Asp Gly Asp 1               5   #                10   #                15 Gly<210> SEQ ID NO 70 <211> LENGTH: 31 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic peptide to asse#s diagnostic potential       of repeat in Lc Gene B <400> SEQUENCE: 70Gly Cys Gly Pro Lys Glu Asp Gly His Thr Gl #n Lys Asn Asp Gly Asp 1               5   #                10   #                15Gly Pro Lys Glu Asp Gly His Thr Gln Lys As #n Asp Gly Asp Gly            20       #            25       #            30<210> SEQ ID NO 71 <211> LENGTH: 45 <212> TYPE: PRT<213> ORGANISM: Gly Cys Gly Pro Lys Glu Asp  #Gly His Thr Gln<220> FEATURE: <223> OTHER INFORMATION: Synthetic peptide to asse#s diagnostic potential       of repeat in Lc Gene B <400> SEQUENCE: 71Gly Cys Gly Pro Lys Glu Asp Gly His Thr Gl #n Lys Asn Asp Gly Asp 1               5   #                10   #                15Gly Pro Lys Glu Asp Gly His Thr Gln Lys As #n Asp Gly Asp Gly Pro            20       #            25       #            30Lys Glu Asp Gly His Thr Gln Lys Asn Asp Gl #y Asp Gly        35           #        40           #        45<210> SEQ ID NO 72 <211> LENGTH: 664 <212> TYPE: DNA<213> ORGANISM: Leishmania major <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(664)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 72gctgcaggaa ttcggcacga gattgcttcc cagcccacct tcgctatcca gc#cactctcg     60ctcttctaca tctcccaccc cctcacaccg ccatggcttc ttcccgcaag gc#ttccaacc    120cgcacaagtc gcaccgcaag ccgaagcgct cgtggaacgt gtacgtgggc cg#ctcgctga    180aggcgatcaa cgcccagatg tcgatgtcgc accgcacgat gaagatcgtg aa#ctcgtacg    240tgaacgacgt gatggagcgc atctgcactg aggccgcgtc gattgttcgc gc#gaacaaga    300agcgcacgtt gggtgcgcgc gaggtgcaga cggcggtgcg cattgtgctg cc#ggcggagc    360tcgcgaagca tgccatggct gagggcacga aggccgtgtc gagcgcgtcc cg#ctaaagcg    420gcttgccgga tgccgtgtga gtaggagggt ggcttgccgc aaacgctgac ct#cggcgatt    480gcggcgtggc gctccccttc tcctccttgt ccggcggtgt gtgtcatgca tt#tgcgtgac    540tcctccctct tatagatgca agcttttttt ttctcttgac gttttatttt ct#cctccccc    600tcccttaacg tgaagtgtat atganagcgt actggacatg ananaaaaaa aa#aanaaact    660 cgag                  #                  #                   #            664 <210> SEQ ID NO 73<211> LENGTH: 1432 <212> TYPE: DNA <213> ORGANISM: Leishmania major<220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)...(1432)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 73gatgaagaag aggaggacac caccatcaac aactccgacg tggtggtgcg ct#acaagaag     60gccgcaacgt ggtgcaatga aacgttgcgc gtgcttatcg atgccacaaa ac#ctggcgcc    120aaggtgtgcg acctgtgccg cctcggtgat gacaccatca ccgccnaggt ca#agacaatg    180ttcaaaggca cggaaaaagg catcgctttc ccgacctgca tctcggtcaa ca#actgcgta    240tgccacaaca gccctggcgt gtcggacgag acgacgcagc aagagatcgc ga#tgggtgac    300gtcgtgcact acgacctggg catccacgtg gacggctact gcgccgtcgt cg#cgcacacc    360attcaggtga cagaggacaa tgagcttggc aaggacgaga aggcggcgcg cg#tcattaca    420gcggcgtaca acatcctgaa cacggcgctg cgccagatgc gtcccggtac ga#ccatctac    480caggtgacag acgtagttga gaaggctgcg gagcactaca aggtgactcc gg#tagacggc    540gtcctctcgc atatgatgaa gcgctacatc atagacngat accgctgtat cc#cgcagcgc    600agggtcgcgg agcacatggt gcacgactac gatctcgaga aagcgcaggt gt#ggacgcta    660gacattgtca tgacctccgg caagggcaag ctgaaggagc gcgatgcgcg gc#cgtgcgtg    720ttcaaggtgg ctctggactc caactactct gtgaaaatgg aaagcgcgaa gg#aggttcag    780aaggaaatcg actccnagta tgccaccttc ccctttgcca tccgcaacct gg#aggccaag    840aaggcccgcc tcggtctcaa cgagatggcg aagcacggtg ctgtcatccc gt#accctatt    900ctcttcgaaa aggaaggcga ggtcgtcgcc catttcaaga ttacggtgct ca#tcagcaac    960aagaagattg agccgattac cggcctgaag ccgcagaagg ccccggcgct cg#agccatac   1020acggacgaga tgctgcttgc gacgaacaag ctcttcgctg tcgctagaga ag#aaggcggc   1080gaagtagacg gccgtggcat ccgtgacgct gtactgcgag ctttcgtagg cg#tacgcctc   1140ttgtgaggcg tacacgtgtg ctgtttgcgg acgaggaggc acccattctg tt#ccccttct   1200tcgctaatct tcgcgtttcc tctgacgctg gcttctytgc cggagtgtgg tg#aggcgcgt   1260gggggagaaa cggcccacty gcatgcctgt gcatacgcga gcacggtagg ga#gcgcggtg   1320tgtgtgtgtg tgggggggcg tgttacgagt acaaaagagg ctcgatcttt gc#gatctttt   1380ctttctgtaa acaggaacat aagtaaccaa aaaaaaaaaa aaaaaactcg ag#           1432 <210> SEQ ID NO 74 <211> LENGTH: 873 <212> TYPE: DNA<213> ORGANISM: Leishmania major <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(873)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 74ctttattgtc atcactgtaa agcactgttt tttctttcac tttttcttga gt#gttttctt     60tattcacca tgagcattat caaggaggac gacgccgtgg gctgctacat gac#ggtgacc     120ctcgtggacg acaccaaggt ggagggtacc atcttcacct acaattccaa gg#agggcatc    180atagtactcc tgtccctccg cgacgatcag acgaacatga agctaatccg ca#ctccgtac    240atcaaagact tcagcctttc acacgctgag gagggagcgc acctgccccc gg#cactggac    300tccttcaacg agcttccgtc catgcacgcc ggccgcgaca agtccatctt ca#agcacgcc    360agcacgcagc tcaagaacgc cgaggcgaac cgcgaaaagc acttcaactc tg#tcacgacc    420gacacaccga ttgccacact tgatgcgtac ctcaagctcc tgcggctata cc#ccttaatt    480gagtggaaca gcgacgaggg tgtcatccag gtctcggaca ccgtcattgt cg#taggagac    540cccgactggc ggacgcccaa ggcaatgctg gtggacggcg cccctgagaa gg#acagaccg    600cttgtagatc gcctgcaggt tgcgctcggm aacggcaaga agtgattcag tg#tgtagcgg    660acagaacatc gtgtgcttgt gtgtctgttt gangtttgtt tgttttctct tt#gtggtact    720gcgtacgacg gcgccttctc ccggtggtgg gtgagtccat aagcagttga gt#tctyggtt    780gtagnaavgc ctyacygccg accatatggg agagggcgaa caaatntttg at#agaagttg    840 aaaatcccaa agtyaaaaga aaaaaaaaan aaa       #                   #        873 <210> SEQ ID NO 75 <211> LENGTH: 1238<212> TYPE: DNA <213> ORGANISM: Leishmania major <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(1238)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 75tttctgtact ttattgaaca tcagtagaac acgttcttcc cgcaaagatg gc#caagaagc     60acctcaagcg cttgtatgcg cccaaggact ggatgctgag caagctgacc gg#cgtgttcg    120cgccgcgtcc gcgtccgggt ccgcacaagc tgcgcgagtg cctgccgctn ct#ggtgatca    180tccgcaaccg gctgaagtac gcgctgaacg cgcgcgaggg tgagatgatc ct#gcgccagg    240gtctggtgca cgtggacaac cacccgcgcc gcgacggcaa gtatcccgcc gg#tttcatgg    300acgtggtcga gatcccgaag acgggcgacc gcttccgcct gatgtacgac gt#caagggcc    360gcttcgcgtt ggtgaacctg tccgaggcgg aggcgcagat caagctgatg aa#ggttgtga    420acctgtacac ggccaccggc cgcgtgccgg tcgctgtgac gcacgacggc ca#ccgcatcc    480gctacccgga cccgcacacc tccattggtg acaccatcgt gtacaacgtc aa#ggagaaga    540agtgcgtgga cctgatcaag aaccgccagg gcaaggccgt gatcgtgacc gg#tggcgcca    600accgcggccg catcggcgag atcgtgaagg tggagtgcca ccccggtgcg tt#caacattg    660cgcacctgaa ggacgcgtcc ggcgccgagt tcgccacccg cgccgcgaac at#cttcgtga    720tcggcaagga cctgaacaac ctgcaggtaa cggtgccgaa gcagcagggc ct#gcgcatga    780acgtgatcca ggagcgcgag gagcgcctga tcgcggcgga ggcccgcaag aa#cgcgccgg    840ctcgtggtgc ccgcagggcc cgcaagtgag gaggcgatta cacgcatgcg tg#tttgtggc    900tctgaagcga cttggcgggt cggctgtgag ggtttgagag gaggtgtgtg at#gcgtgtga    960agtccttctc cgttctcagc tctctctgtg ctgtagctgt gcctttcccc ag#atcgcttt   1020accgcatttg catacatctg tgtagtcgca tgtgcgtgtt tctgtctctc gg#tgggtctc   1080cctctccctc cctttctgcc tctctctttg agtgggtgtg catgcgtcgc gc#gcgacggg   1140ctccgcttna gtgattctct cgtgttttan ggctgtttty tttctyagtt na#gcgtttty   1200 gttcatgatt tcctcagacc caaaaaaaaa aaaaaaaa      #                   #   1238 210> SEQ ID NO 76 <211> LENGTH: 712<212> TYPE: DNA <213> ORGANISM: Leishmania major <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(712)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 76ctgacggagt tccagacgaa ccttgtgccg tacccgcgca tccacttcgt gc#tgacaagc     60tacgctccgg tggtgtctgc cgagaaggcg taccacgagc agctntccgt cg#cggacatc    120acgaactcgg tntttgagcc tgctggcatg ctnacaaagt gcgatcctcg cc#acggcaag    180tacatgtcgt gctgcctcat gtaccgcggt gatgtcgtgc cgaaggatgt ca#acgccgcg    240attgcgacga tcaagacgaa gcgcacaatt cagttcgtgg actggtgccc ga#ccggcttc    300aagtgcggca tcaactacca gccgccgacc gttgtgcccg gcggtgacct cg#cgaaggtg    360cagcgcgccg tgtgcatgat tgccaactcg accgcgatcg ctgaggtgtt tg#cccgcatc    420gaccacaagt tcgacctgat gtacagcaag cgcgcgtttg tgcactggta cg#tgggtgag    480ggcatggagg agggcgagtt ctccgaggcg cgcgaggatc tcgctgcgct gg#agaaggac    540tacgaggagg ttggcgccga gtccgccgac gacatgggcg aggaggacgt cg#aggagtac    600taaggtagac tcgtgccgcg cgctgatgat gtaggtgcac gcgtgcgtgt gc#tgcagcgg    660agccgccgcc accgcgactg tgtgtgtgtg cgcgcgtgac gaccggctcg ag#            712 <210> SEQ ID NO 77 <211> LENGTH: 1086 <212> TYPE: DNA<213> ORGANISM: Leishmania major <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(1086)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 77caagaagtgg atcaagcagg agacgaacgc cgatggcgag cgcgtgcgcc gc#gcgttctg     60ccagttctgc ctagacccca tctaccagat cttcgacgct gtgatgaacg ag#aagaagga    120caaggtggac aagatgctca agtcgctgca cgtgacgctn acggctgagg ag#cgcgagca    180ggtgccgaan aagcttctga agacggtgat gatgaanttc ctgccggctg ct#gagacgct    240gctacagatg atcgtggcgc acctgccgtc gcccaagaag gcgcaggcgt ac#cgtgcgga    300gatgctgtac tctggcgagg cgtcgccgga ggacaagtac ttcatgggta tc#aagaactg    360cgaccccgct gcgccgctca tgctgtacat cagcaagatg gtgccgacgg cc#gaccgcgg    420ccgcttcttc gcctttggcc gcatcttctc cggtaaggtg cgcagcggcc ag#aaggtgcg    480catcatgggt aacaactacg tctacggcaa gaagcaggac ctgtacgagg ac#aagcctgt    540gcagcgctcc gtgctgatga tgggccgcta ccaggaggcc gtggaggaca tg#ccgtgcgg    600taacgtggtg ggccttgtgg gcgtggacaa gtacatcgtg aagtccgcga cg#atcacgga    660cgatggcgag agcccgcacc cgctgcgcga catgaagtac tctgtgtcgc cc#gtcgtgcg    720tgtggccgtg gaggcgaaga acccgtccga cctgccgaag cttgtggagg gc#ctgaagcg    780ccttgccaag tccgacccgc tggtggtgtg cagcattgag gagtctggcg ag#cacattgt    840tgccggcgct ggcgagcttc accttgagat ttgcctgaag gatctccagg ag#gacttcat    900gaacggcgcg ccgctnaaga tctccgagcc ggtggtgtcg ttccgcgaga cg#gtgacgga    960tgtgtcgtcg cagcagtgcc tgtcgaagtc tgcgaacaag cacaaccgtc tc#ttctgccg   1020cggtgcgccg ctnacagagg anctggcgct ggcgatngan gaaggcaccg ct#ggtcccga   1080 ngcgga                  #                  #                   #         1086 <210> SEQ ID NO 78 <211> LENGTH: 447<212> TYPE: DNA <213> ORGANISM: Leishmania major <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(447)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 78cgcatcaacg tctacttcga tnagtcgacg ggaggccgct acgtgccgcg cg#ccgtgctg     60atggacctcg agcccggcac tatggactcc gttcgcgccg gcccgtacgg cc#agctgttc    120cgcccggaca acttcatctt tggtcagtcc ggcgctggca acaactgggc ca#agggccac    180tacactgagg gcgcggagct gatcgactcc gtgcttgatg tgtgccgcaa gg#aggcggag    240agctgcgact gcctgcaggg cttccagctg tctcactccc tcggcggcgg ca#cgggctcc    300ggcatgggca cgctgctcat ttccaanctg cgcgangagt acccggaccg ga#tcatgatg    360accttctccg tcatcccgtc cccccgcgtg tcggataccg ttgtggancc gt#acaacacg    420 accctctctg tgcaccagct cgtggaa          #                   #            447 <210> SEQ ID NO 79<211> LENGTH: 375 <212> TYPE: DNA <213> ORGANISM: Leishmania major<220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)...(375)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 79gtaacccgct ggtgtacgca tatgtagaca cagacgggca gcacgagacg ac#gttcctcg     60cgatccctgt ggtgcttggc atgaatggaa tcgagaagcg cctgccgatt gg#tccgctgc    120actcgacgga ggaaacgctg ctgaaggcgg cactgccggt gatcaagaag aa#tatcgtga    180agggcagcga gttcgcgcgc tcacacctgt agcacctcag cttttttttt tt#gcgttaaa    240cgggcgtggg aagcacctcg atacttcgct tcgcgctgac ggacccgcac ga#catcgttc    300gtcatccccc tccccctctt cggccctata cgcatgaagg agtggaatta tg#caacagca    360 tgttnatatc aagtg               #                  #                   #   375 <210> SEQ ID NO 80 <211> LENGTH: 107<212> TYPE: PRT <213> ORGANISM: Leishmania major <400> SEQUENCE: 80Met Ala Ser Ser Arg Lys Ala Ser Asn Pro Hi #s Lys Ser His Arg Lys 1               5   #                10   #                15Pro Lys Arg Ser Trp Asn Val Tyr Val Gly Ar #g Ser Leu Lys Ala Ile            20       #            25       #            30Asn Ala Gln Met Ser Met Ser His Arg Thr Me #t Lys Ile Val Asn Ser        35           #        40           #        45Tyr Val Asn Asp Val Met Glu Arg Ile Cys Th #r Glu Ala Ala Ser Ile    50               #    55               #    60Val Arg Ala Asn Lys Lys Arg Thr Leu Gly Al #a Arg Glu Val Gln Thr65                   #70                   #75                   #80Ala Val Arg Ile Val Leu Pro Ala Glu Leu Al #a Lys His Ala Met Ala                85   #                90   #                95Glu Gly Thr Lys Ala Val Ser Ser Ala Ser Ar #g             100      #           105 <210> SEQ ID NO 81 <211> LENGTH: 381 <212> TYPE: PRT<213> ORGANISM: Leishmania major <220> FEATURE: <221> NAME/KEY: VARIANT<222> LOCATION: (1)...(381) <223> OTHER INFORMATION: Xaa = Any Amino Aci#d <400> SEQUENCE: 81 Asp Glu Glu Glu Glu Asp Thr Thr Ile Asn As#n Ser Asp Val Val Val  1               5   #                10  #                15 Arg Tyr Lys Lys Ala Ala Thr Trp Cys Asn Gl#u Thr Leu Arg Val Leu             20       #            25      #            30 Ile Asp Ala Thr Lys Pro Gly Ala Lys Val Cy#s Asp Leu Cys Arg Leu         35           #        40          #        45 Gly Asp Asp Thr Ile Thr Ala Xaa Val Lys Th#r Met Phe Lys Gly Thr     50               #    55              #    60 Glu Lys Gly Ile Ala Phe Pro Thr Cys Ile Se#r Val Asn Asn Cys Val 65                   #70                  #75                   #80 Cys His Asn Ser Pro Gly Val Ser Asp Glu Th#r Thr Gln Gln Glu Ile                 85   #                90  #                95 Ala Met Gly Asp Val Val His Tyr Asp Leu Gl#y Ile His Val Asp Gly             100       #           105      #           110 Tyr Cys Ala Val Val Ala His Thr Ile Gln Va#l Thr Glu Asp Asn Glu         115           #       120          #       125 Leu Gly Lys Asp Glu Lys Ala Ala Arg Val Il#e Thr Ala Ala Tyr Asn     130               #   135              #   140 Ile Leu Asn Thr Ala Leu Arg Gln Met Arg Pr#o Gly Thr Thr Ile Tyr 145                 1 #50                 1#55                 1 #60 Gln Val Thr Asp Val Val Glu Lys Ala Ala Gl#u His Tyr Lys Val Thr                 165   #               170  #               175 Pro Val Asp Gly Val Leu Ser His Met Met Ly#s Arg Tyr Ile Ile Asp             180       #           185      #           190 Xaa Tyr Arg Cys Ile Pro Gln Arg Arg Val Al#a Glu His Met Val His         195           #       200          #       205 Asp Tyr Asp Leu Glu Lys Ala Gln Val Trp Th#r Leu Asp Ile Val Met     210               #   215              #   220 Thr Ser Gly Lys Gly Lys Leu Lys Glu Arg As#p Ala Arg Pro Cys Val 225                 2 #30                 2#35                 2 #40 Phe Lys Val Ala Leu Asp Ser Asn Tyr Ser Va#l Lys Met Glu Ser Ala                 245   #               250  #               255 Lys Glu Val Gln Lys Glu Ile Asp Ser Xaa Ty#r Ala Thr Phe Pro Phe             260       #           265      #           270 Ala Ile Arg Asn Leu Glu Ala Lys Lys Ala Ar#g Leu Gly Leu Asn Glu         275           #       280          #       285 Met Ala Lys His Gly Ala Val Ile Pro Tyr Pr#o Ile Leu Phe Glu Lys     290               #   295              #   300 Glu Gly Glu Val Val Ala His Phe Lys Ile Th#r Val Leu Ile Ser Asn 305                 3 #10                 3#15                 3 #20 Lys Lys Ile Glu Pro Ile Thr Gly Leu Lys Pr#o Gln Lys Ala Pro Ala                 325   #               330  #               335 Leu Glu Pro Tyr Thr Asp Glu Met Leu Leu Al#a Thr Asn Lys Leu Phe             340       #           345      #           350 Ala Val Ala Arg Glu Glu Gly Gly Glu Val As#p Gly Arg Gly Ile Arg         355           #       360          #       365 Asp Ala Val Leu Arg Ala Phe Val Gly Val Ar #g Leu Leu    370               #   375               #   380 <210> SEQ ID NO 82<211> LENGTH: 191 <212> TYPE: PRT <213> ORGANISM: Leishmania major<400> SEQUENCE: 82 Met Ser Ile Ile Lys Glu Asp Asp Ala Val Gl#y Cys Tyr Met Thr Val  1               5   #                10  #                15 Thr Leu Val Asp Asp Thr Lys Val Glu Gly Th#r Ile Phe Thr Tyr Asn             20       #            25      #            30 Ser Lys Glu Gly Ile Ile Val Leu Leu Ser Le#u Arg Asp Asp Gln Thr         35           #        40          #        45 Asn Met Lys Leu Ile Arg Thr Pro Tyr Ile Ly#s Asp Phe Ser Leu Ser     50               #    55              #    60 His Ala Glu Glu Gly Ala His Leu Pro Pro Al#a Leu Asp Ser Phe Asn 65                   #70                  #75                   #80 Glu Leu Pro Ser Met His Ala Gly Arg Asp Ly#s Ser Ile Phe Lys His                 85   #                90  #                95 Ala Ser Thr Gln Leu Lys Asn Ala Glu Ala As#n Arg Glu Lys His Phe             100       #           105      #           110 Asn Ser Val Thr Thr Asp Thr Pro Ile Ala Th#r Leu Asp Ala Tyr Leu         115           #       120          #       125 Lys Leu Leu Arg Leu Tyr Pro Leu Ile Glu Tr#p Asn Ser Asp Glu Gly     130               #   135              #   140 Val Ile Gln Val Ser Asp Thr Val Ile Val Va#l Gly Asp Pro Asp Trp 145                 1 #50                 1#55                 1 #60 Arg Thr Pro Lys Ala Met Leu Val Asp Gly Al#a Pro Glu Lys Asp Arg                 165   #               170  #               175 Pro Leu Val Asp Arg Leu Gln Val Ala Leu Gl#y Asn Gly Lys Lys             180       #           185      #           190 <210> SEQ ID NO 83 <211> LENGTH: 273 <212> TYPE: PRT<213> ORGANISM: Leishmania major <400> SEQUENCE: 83Met Ala Lys Lys His Leu Lys Arg Leu Tyr Al #a Pro Lys Asp Trp Met 1               5   #                10   #                15Leu Ser Lys Leu Thr Gly Val Phe Ala Pro Ar #g Pro Arg Pro Gly Pro            20       #            25       #            30His Lys Leu Arg Glu Cys Leu Pro Leu Leu Va #l Ile Ile Arg Asn Arg        35           #        40           #        45Leu Lys Tyr Ala Leu Asn Ala Arg Glu Gly Gl #u Met Ile Leu Arg Gln    50               #    55               #    60Gly Leu Val His Val Asp Asn His Pro Arg Ar #g Asp Gly Lys Tyr Pro65                   #70                   #75                   #80Ala Gly Phe Met Asp Val Val Glu Ile Pro Ly #s Thr Gly Asp Arg Phe                85   #                90   #                95Arg Leu Met Tyr Asp Val Lys Gly Arg Phe Al #a Leu Val Asn Leu Ser            100       #           105       #           110Glu Ala Glu Ala Gln Ile Lys Leu Met Lys Va #l Val Asn Leu Tyr Thr        115           #       120           #       125Ala Thr Gly Arg Val Pro Val Ala Val Thr Hi #s Asp Gly His Arg Ile    130               #   135               #   140Arg Tyr Pro Asp Pro His Thr Ser Ile Gly As #p Thr Ile Val Tyr Asn145                 1 #50                 1 #55                 1 #60Val Lys Glu Lys Lys Cys Val Asp Leu Ile Ly #s Asn Arg Gln Gly Lys                165   #               170   #               175Ala Val Ile Val Thr Gly Gly Ala Asn Arg Gl #y Arg Ile Gly Glu Ile            180       #           185       #           190Val Lys Val Glu Cys His Pro Gly Ala Phe As #n Ile Ala His Leu Lys        195           #       200           #       205Asp Ala Ser Gly Ala Glu Phe Ala Thr Arg Al #a Ala Asn Ile Phe Val    210               #   215               #   220Ile Gly Lys Asp Leu Asn Asn Leu Gln Val Th #r Val Pro Lys Gln Gln225                 2 #30                 2 #35                 2 #40Gly Leu Arg Met Asn Val Ile Gln Glu Arg Gl #u Glu Arg Leu Ile Ala                245   #               250   #               255Ala Glu Ala Arg Lys Asn Ala Pro Ala Arg Gl #y Ala Arg Arg Ala Arg            260       #           265       #           270 Lys<210> SEQ ID NO 84 <211> LENGTH: 200 <212> TYPE: PRT<213> ORGANISM: Leishmania major <400> SEQUENCE: 84Leu Thr Glu Phe Gln Thr Asn Leu Val Pro Ty #r Pro Arg Ile His Phe 1               5   #                10   #                15Val Leu Thr Ser Tyr Ala Pro Val Val Ser Al #a Glu Lys Ala Tyr His            20       #            25       #            30Glu Gln Leu Ser Val Ala Asp Ile Thr Asn Se #r Val Phe Glu Pro Ala        35           #        40           #        45Gly Met Leu Thr Lys Cys Asp Pro Arg His Gl #y Lys Tyr Met Ser Cys    50               #    55               #    60Cys Leu Met Tyr Arg Gly Asp Val Val Pro Ly #s Asp Val Asn Ala Ala65                   #70                   #75                   #80Ile Ala Thr Ile Lys Thr Lys Arg Thr Ile Gl #n Phe Val Asp Trp Cys                85   #                90   #                95Pro Thr Gly Phe Lys Cys Gly Ile Asn Tyr Gl #n Pro Pro Thr Val Val            100       #           105       #           110Pro Gly Gly Asp Leu Ala Lys Val Gln Arg Al #a Val Cys Met Ile Ala        115           #       120           #       125Asn Ser Thr Ala Ile Ala Glu Val Phe Ala Ar #g Ile Asp His Lys Phe    130               #   135               #   140Asp Leu Met Tyr Ser Lys Arg Ala Phe Val Hi #s Trp Tyr Val Gly Glu145                 1 #50                 1 #55                 1 #60Gly Met Glu Glu Gly Glu Phe Ser Glu Ala Ar #g Glu Asp Leu Ala Ala                165   #               170   #               175Leu Glu Lys Asp Tyr Glu Glu Val Gly Ala Gl #u Ser Ala Asp Asp Met            180       #           185       #           190Gly Glu Glu Asp Val Glu Glu Tyr         195           #       200<210> SEQ ID NO 85 <211> LENGTH: 361 <212> TYPE: PRT<213> ORGANISM: Leishmania major <220> FEATURE: <221> NAME/KEY: VARIANT<222> LOCATION: (1)...(361) <223> OTHER INFORMATION: Xaa = Any Amino Aci#d <400> SEQUENCE: 85 Lys Lys Trp Ile Lys Gln Glu Thr Asn Ala As#p Gly Glu Arg Val Arg  1               5   #                10  #                15 Arg Ala Phe Cys Gln Phe Cys Leu Asp Pro Il#e Tyr Gln Ile Phe Asp             20       #            25      #            30 Ala Val Met Asn Glu Lys Lys Asp Lys Val As#p Lys Met Leu Lys Ser         35           #        40          #        45 Leu His Val Thr Leu Thr Ala Glu Glu Arg Gl#u Gln Val Pro Xaa Lys     50               #    55              #    60 Leu Leu Lys Thr Val Met Met Xaa Phe Leu Pr#o Ala Ala Glu Thr Leu 65                   #70                  #75                   #80 Leu Gln Met Ile Val Ala His Leu Pro Ser Pr#o Lys Lys Ala Gln Ala                 85   #                90  #                95 Tyr Arg Ala Glu Met Leu Tyr Ser Gly Glu Al#a Ser Pro Glu Asp Lys             100       #           105      #           110 Tyr Phe Met Gly Ile Lys Asn Cys Asp Pro Al#a Ala Pro Leu Met Leu         115           #       120          #       125 Tyr Ile Ser Lys Met Val Pro Thr Ala Asp Ar#g Gly Arg Phe Phe Ala     130               #   135              #   140 Phe Gly Arg Ile Phe Ser Gly Lys Val Arg Se#r Gly Gln Lys Val Arg 145                 1 #50                 1#55                 1 #60 Ile Met Gly Asn Asn Tyr Val Tyr Gly Lys Ly#s Gln Asp Leu Tyr Glu                 165   #               170  #               175 Asp Lys Pro Val Gln Arg Ser Val Leu Met Me#t Gly Arg Tyr Gln Glu             180       #           185      #           190 Ala Val Glu Asp Met Pro Cys Gly Asn Val Va#l Gly Leu Val Gly Val         195           #       200          #       205 Asp Lys Tyr Ile Val Lys Ser Ala Thr Ile Th#r Asp Asp Gly Glu Ser     210               #   215              #   220 Pro His Pro Leu Arg Asp Met Lys Tyr Ser Va#l Ser Pro Val Val Arg 225                 2 #30                 2#35                 2 #40 Val Ala Val Glu Ala Lys Asn Pro Ser Asp Le#u Pro Lys Leu Val Glu                 245   #               250  #               255 Gly Leu Lys Arg Leu Ala Lys Ser Asp Pro Le#u Val Val Cys Ser Ile             260       #           265      #           270 Glu Glu Ser Gly Glu His Ile Val Ala Gly Al#a Gly Glu Leu His Leu         275           #       280          #       285 Glu Ile Cys Leu Lys Asp Leu Gln Glu Asp Ph#e Met Asn Gly Ala Pro     290               #   295              #   300 Leu Lys Ile Ser Glu Pro Val Val Ser Phe Ar#g Glu Thr Val Thr Asp 305                 3 #10                 3#15                 3 #20 Val Ser Ser Gln Gln Cys Leu Ser Lys Ser Al#a Asn Lys His Asn Arg                 325   #               330  #               335 Leu Phe Cys Arg Gly Ala Pro Leu Thr Glu Xa#a Leu Ala Leu Ala Xaa             340       #           345      #           350 Xaa Glu Gly Thr Ala Gly Pro Xaa Ala        355           #       360 <210> SEQ ID NO 86 <211> LENGTH: 149<212> TYPE: PRT <213> ORGANISM: Leishmania major <220> FEATURE:<221> NAME/KEY: VARIANT <222> LOCATION: (1)...(149)<223> OTHER INFORMATION: Xaa = Any Amino Aci #d <400> SEQUENCE: 86Arg Ile Asn Val Tyr Phe Asp Xaa Ser Thr Gl #y Gly Arg Tyr Val Pro 1               5   #                10   #                15Arg Ala Val Leu Met Asp Leu Glu Pro Gly Th #r Met Asp Ser Val Arg            20       #            25       #            30Ala Gly Pro Tyr Gly Gln Leu Phe Arg Pro As #p Asn Phe Ile Phe Gly        35           #        40           #        45Gln Ser Gly Ala Gly Asn Asn Trp Ala Lys Gl #y His Tyr Thr Glu Gly    50               #    55               #    60Ala Glu Leu Ile Asp Ser Val Leu Asp Val Cy #s Arg Lys Glu Ala Glu65                   #70                   #75                   #80Ser Cys Asp Cys Leu Gln Gly Phe Gln Leu Se #r His Ser Leu Gly Gly                85   #                90   #                95Gly Thr Gly Ser Gly Met Gly Thr Leu Leu Il #e Ser Xaa Leu Arg Xaa            100       #           105       #           110Glu Tyr Pro Asp Arg Ile Met Met Thr Phe Se #r Val Ile Pro Ser Pro        115           #       120           #       125Arg Val Ser Asp Thr Val Val Xaa Pro Tyr As #n Thr Thr Leu Ser Val    130               #   135               #   140 His Gln Leu Val Glu145 <210> SEQ ID NO 87 <211> LENGTH: 69 <212> TYPE: PRT<213> ORGANISM: Leishmania major <400> SEQUENCE: 87Asn Pro Leu Val Tyr Ala Tyr Val Asp Thr As #p Gly Gln His Glu Thr 1               5   #                10   #                15Thr Phe Leu Ala Ile Pro Val Val Leu Gly Me #t Asn Gly Ile Glu Lys            20       #            25       #            30Arg Leu Pro Ile Gly Pro Leu His Ser Thr Gl #u Glu Thr Leu Leu Lys        35           #        40           #        45Ala Ala Leu Pro Val Ile Lys Lys Asn Ile Va #l Lys Gly Ser Glu Phe    50               #    55               #    60 Ala Arg Ser His Leu65 <210> SEQ ID NO 88 <211> LENGTH: 54 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: PCR primer <400> SEQUENCE: 88agtattcata tgcaccacca ccaccaccac atgtcctgcg gtaacgccaa ga#tc           54 <210> SEQ ID NO 89 <211> LENGTH: 33 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: PCR primer <400> SEQUENCE: 89ctcacaggat ccctgcttgc tgaagtatcc ttc        #                  #         33 <210> SEQ ID NO 90 <211> LENGTH: 36 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: PCR primer <400> SEQUENCE: 90catttcggat ccatggacgc aactgagctg aagaac       #                  #       36 <210> SEQ ID NO 91 <211> LENGTH: 33 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: PCR primer <400> SEQUENCE: 91cgtagagaat tcctgaccaa aacgaatgat gcc        #                  #         33 <210> SEQ ID NO 92 <211> LENGTH: 33 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: PCR primer <400> SEQUENCE: 92caccacgaat tcatggcgca gaatgataag atc        #                  #         33 <210> SEQ ID NO 93 <211> LENGTH: 34 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: PCR primer <400> SEQUENCE: 93actgacctcg aggaattctt agtcgcgcat gaac        #                  #        34 <210> SEQ ID NO 94 <211> LENGTH: 3012 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: DNA sequence encoding fus #ion (poly-protein)      constructs comprising multiple Leishman #ia antigens<400> SEQUENCE: 94catatgcacc accaccacca ccacatgtcc tgcggtaacg ccaagatcaa ct#ctcccgcg     60ccgtccttcg aggaggtggc gctcatgccc aacggcagct tcaagaagat ca#gcctctcc    120tcctacaagg gcaagtgggt cgtgctcttc ttctacccgc tcgacttcac ct#tcgtgtgc    180ccgacagagg tcatcgcgtt ctccgacagc gtgagtcgct tcaacgagct ca#actgcgag    240gtcctcgcgt gctcgataga cagcgagtac gcgcacctgc agtggacgct gc#aggaccgc    300aagaagggcg gcctcgggac catggcgatc ccaatgctag ccgacaagac ca#agagcatc    360gctcgttcct acggcgtgct ggaggagagc cagggcgtgg cctaccgcgg tc#tcttcatc    420atcgaccccc atggcatgct gcgtcagatc accgtcaatg acatgccggt gg#gccgcagc    480gtggaggagg ttctacgcct gctggaggct tttcagttcg tggagaagca cg#gcgaggtg    540tgccccgcga actggaagaa gggcgccccc acgatgaagc cggaaccgaa tg#cgtctgtc    600gagggatact tcagcaagca gggatccatg gacgcaactg agctgaagaa ca#aggggaac    660gaagagttct ccgccggccg ctatgtggag gcggtgaact acttctcaaa gg#cgatccag    720ttggatgagc agaacagtgt cctctacagc aaccgctccg cctgttttgc ag#ccatgcag    780aaatacaagg acgcgctgga cgacgccgac aagtgcatct cgatcaagcc ga#attgggcc    840aagggctacg tgcgccgagg agcagctctc catggcatgc gccgctacga cg#atgccatt    900gccgcgtatg aaaaggggct caaggtggac ccttccaaca gcggctgcgc gc#agggcgtg    960aaggacgtgc aggtagccaa ggcccgcgaa gcacgtgacc ccatcgctcg cg#tcttcacc   1020ccggaggcgt tccgcaagat ccaagagaat cccaagctgt ctctacttat gc#tgcagccg   1080gactacgtga agatggtaga caccgtcatc cgcgaccctt cgcagggccg gc#tgtacatg   1140gaagaccagc gctttgccct gacgctcatg tacctgagcg gaatgaagat tc#ccaacgat   1200ggtgatggcg aggaggagga acgtccgtct gcgaaggcgg cagagacagc ga#agccaaaa   1260gaggagaagc ctctcaccga caacgagaag gaggccctgg cgctcaagga gg#agggcaac   1320aagctgtacc tctcgaagaa gtttgaggag gcgctgacca agtaccaaga gg#cgcaggtg   1380aaagacccca acaacacttt atacattctg aacgtgtcgg ccgtgtactt cg#agcagggt   1440gactacgaca agtgcatcgc cgagtgcgag cacggtatcg agcacggtcg cg#agaaccac   1500tgcgactaca caatcattgc gaagctcatg acccggaacg ccttgtgcct cc#agaggcag   1560aggaagtacg aggctgctat cgacctttac aagcgcgccc ttgtcgagtg gc#gtaaccct   1620gacaccctca agaagctgac ggagtgcgag aaggagcacc aaaaggcggt gg#aggaagcc   1680tacatcgatc ctgagatcgc gaagcagaag aaagacgaag gtaaccagta ct#tcaaggag   1740gataagttcc ccgaggccgt ggcagcgtac acggaggcca tcaagcgcaa cc#ctgccgag   1800cacacctcct acagcaatcg cgcggccgcg tacatcaagc ttggagcctt ca#acgacgcc   1860ctcaaggacg cggagaagtg cattgagctg aagcccgact ttgttaaggg ct#acgcgcgc   1920aagggtcatg cttacttttg gaccaagcag tacaaccgcg cgctgcaggc gt#acaatgag   1980ggcctcaagg tggacccgag caatgcggac tgcaaggatg ggcggtatcg ca#caatcatg   2040aagattcagg agatggcatc tggccaatcc gcggatggcg acgaggcggc gc#gccgggcc   2100atggacgatc ctgaaatcgc ggcaatcatg caagatagct acatgcaact ag#tgttgaag   2160gagatgcaga acgatcccac gcgcattcag gagtacatga aggactccgg ga#tctcatcg   2220aagatcaaca agctgatttc agctggcatc attcgttttg gtcaggaatt ca#tggcgcag   2280aatgataaga tcgcccccca ggaccaggac tccttcctcg atgaccagcc cg#gcgttcgc   2340ccgatcccgt ccttcgacga catgccgctg caccagaacc tgctgcgtgg ca#tctactcg   2400tacgggttcg agaagccgtc cagcatccag cagcgcgcga tagccccctt ca#cgcgcggc   2460ggcgacatca tcgcgcaggc ccagtccggt accggcaaga cgggtgcctt ct#ccatcggt   2520ctgctgcagc gcctggactt ccgccacaac ctgatccagg gcctcgtgct ct#cccccact   2580cgcgagctgg ccctgcagac ggcggaggtg atcagccgca tcggtgagtt cc#tgtcgaac   2640agctccaagt tctgcgagac ctttgtcggc ggcacgcgcg tgcaggatga cc#tgcgcaag   2700ctgcaggccg gcgtcatcgt tgccgtgggc acgccgggcc gcgtgtccga cg#tgatcaag   2760cgtggcgcgc tgcgcacaga gtcgctgcgc gtgctggtgc tcgacgaggc tg#atgagatg   2820ctgtctcagg gcttcgcgga ccagatttac gagatcttcc gcttcctgcc ga#aggacatc   2880caggtcgcgc tcttctccgc cacgatgccg gaggaggtac tggagctgac ga#agaagttc   2940atgcgcgact aagaattcct cgagcagatc cggctgctaa caaagcccga aa#ggaagctg   3000 aatggctgct gc               #                  #                   #     3012 <210> SEQ ID NO 95 <211> LENGTH: 982<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Fusion (poly-protein) constr #ucts comprising      multiple Leishmania antigens <400> SEQUENCE: 95Met His His His His His His Met Ser Cys Gl #y Asn Ala Lys Ile Asn            5       #            10       #            15Ser Pro Ala Pro Ser Phe Glu Glu Val Ala Le #u Met Pro Asn Gly Ser             20      #             25      #             30Phe Lys Lys Ile Ser Leu Ser Ser Tyr Lys Gl #y Lys Trp Val Val Leu         35          #         40          #         45Phe Phe Tyr Pro Leu Asp Phe Thr Phe Val Cy #s Pro Thr Glu Val Ile     50              #     55              #     60Ala Phe Ser Asp Ser Val Ser Arg Phe Asn Gl #u Leu Asn Cys Glu Val 65                  # 70                  # 75                  # 80Leu Ala Cys Ser Ile Asp Ser Glu Tyr Ala Hi #s Leu Gln Trp Thr Leu                 85  #                 90  #                 95Gln Asp Arg Lys Lys Gly Gly Leu Gly Thr Me #t Ala Ile Pro Met Leu            100       #           105       #           110Ala Asp Lys Thr Lys Ser Ile Ala Arg Ser Ty #r Gly Val Leu Glu Glu        115           #       120           #       125Ser Gln Gly Val Ala Tyr Arg Gly Leu Phe Il #e Ile Asp Pro His Gly    130               #   135               #   140Met Leu Arg Gln Ile Thr Val Asn Asp Met Pr #o Val Gly Arg Ser Val145                 1 #50                 1 #55                 1 #60Glu Glu Val Leu Arg Leu Leu Glu Ala Phe Gl #n Phe Val Glu Lys His                165   #               170   #               175Gly Glu Val Cys Pro Ala Asn Trp Lys Lys Gl #y Ala Pro Thr Met Lys            180       #           185       #           190Pro Glu Pro Asn Ala Ser Val Glu Gly Tyr Ph #e Ser Lys Gln Gly Ser        195           #       200           #       205Met Asp Ala Thr Glu Leu Lys Asn Lys Gly As #n Glu Glu Phe Ser Ala    210               #   215               #   220Gly Arg Tyr Val Glu Ala Val Asn Tyr Phe Se #r Lys Ala Ile Gln Leu225                 2 #30                 2 #35                 2 #40Asp Glu Gln Asn Ser Val Leu Tyr Ser Asn Ar #g Ser Ala Cys Phe Ala                245   #               250   #               255Ala Met Gln Lys Tyr Lys Asp Ala Leu Asp As #p Ala Asp Lys Cys Ile            260       #           265       #           270Ser Ile Lys Pro Asn Trp Ala Lys Gly Tyr Va #l Arg Arg Gly Ala Ala        275           #       280           #       285Leu His Gly Met Arg Arg Tyr Asp Asp Ala Il #e Ala Ala Tyr Glu Lys    290               #   295               #   300Gly Leu Lys Val Asp Pro Ser Asn Ser Gly Cy #s Ala Gln Gly Val Lys305                 3 #10                 3 #15                 3 #20Asp Val Gln Val Ala Lys Ala Arg Glu Ala Ar #g Asp Pro Ile Ala Arg                325   #               330   #               335Val Phe Thr Pro Glu Ala Phe Arg Lys Ile Gl #n Glu Asn Pro Lys Leu            340       #           345       #           350Ser Leu Leu Met Leu Gln Pro Asp Tyr Val Ly #s Met Val Asp Thr Val        355           #       360           #       365Ile Arg Asp Pro Ser Gln Gly Arg Leu Tyr Me #t Glu Asp Gln Arg Phe    370               #   375               #   380Ala Leu Thr Leu Met Tyr Leu Ser Gly Met Ly #s Ile Pro Asn Asp Gly385                 3 #90                 3 #95                 4 #00Asp Gly Glu Glu Glu Glu Arg Pro Ser Ala Ly #s Ala Ala Glu Thr Ala                405   #               410   #               415Lys Pro Lys Glu Glu Lys Pro Leu Thr Asp As #n Glu Lys Glu Ala Leu            420       #           425       #           430Ala Leu Lys Glu Glu Gly Asn Lys Leu Tyr Le #u Ser Lys Lys Phe Glu        435           #       440           #       445Glu Ala Leu Thr Lys Tyr Gln Glu Ala Gln Va #l Lys Asp Pro Asn Asn    450               #   455               #   460Thr Leu Tyr Ile Leu Asn Val Ser Ala Val Ty #r Phe Glu Gln Gly Asp465                 4 #70                 4 #75                 4 #80Tyr Asp Lys Cys Ile Ala Glu Cys Glu His Gl #y Ile Glu His Gly Arg                485   #               490   #               495Glu Asn His Cys Asp Tyr Thr Ile Ile Ala Ly #s Leu Met Thr Arg Asn            500       #           505       #           510Ala Leu Cys Leu Gln Arg Gln Arg Lys Tyr Gl #u Ala Ala Ile Asp Leu        515           #       520           #       525Tyr Lys Arg Ala Leu Val Glu Trp Arg Asn Pr #o Asp Thr Leu Lys Lys    530               #   535               #   540Leu Thr Glu Cys Glu Lys Glu His Gln Lys Al #a Val Glu Glu Ala Tyr545                 5 #50                 5 #55                 5 #60Ile Asp Pro Glu Ile Ala Lys Gln Lys Lys As #p Glu Gly Asn Gln Tyr                565   #               570   #               575Phe Lys Glu Asp Lys Phe Pro Glu Ala Val Al #a Ala Tyr Thr Glu Ala            580       #           585       #           590Ile Lys Arg Asn Pro Ala Glu His Thr Ser Ty #r Ser Asn Arg Ala Ala        595           #       600           #       605Ala Tyr Ile Lys Leu Gly Ala Phe Asn Asp Al #a Leu Lys Asp Ala Glu    610               #   615               #   620Lys Cys Ile Glu Leu Lys Pro Asp Phe Val Ly #s Gly Tyr Ala Arg Lys625                 6 #30                 6 #35                 6 #40Gly His Ala Tyr Phe Trp Thr Lys Gln Tyr As #n Arg Ala Leu Gln Ala                645   #               650   #               655Tyr Asn Glu Gly Leu Lys Val Asp Pro Ser As #n Ala Asp Cys Lys Asp            660       #           665       #           670Gly Arg Tyr Arg Thr Ile Met Lys Ile Gln Gl #u Met Ala Ser Gly Gln        675           #       680           #       685Ser Ala Asp Gly Asp Glu Ala Ala Arg Arg Al #a Met Asp Asp Pro Glu    690               #   695               #   700Ile Ala Ala Ile Met Gln Asp Ser Tyr Met Gl #n Leu Val Leu Lys Glu705                 7 #10                 7 #15                 7 #20Met Gln Asn Asp Pro Thr Arg Ile Gln Glu Ty #r Met Lys Asp Ser Gly                725   #               730   #               735Ile Ser Ser Lys Ile Asn Lys Leu Ile Ser Al #a Gly Ile Ile Arg Phe            740       #           745       #           750Gly Gln Glu Phe Met Ala Gln Asn Asp Lys Il #e Ala Pro Gln Asp Gln        755           #       760           #       765Asp Ser Phe Leu Asp Asp Gln Pro Gly Val Ar #g Pro Ile Pro Ser Phe    770               #   775               #   780Asp Asp Met Pro Leu His Gln Asn Leu Leu Ar #g Gly Ile Tyr Ser Tyr785                 7 #90                 7 #95                 8 #00Gly Phe Glu Lys Pro Ser Ser Ile Gln Gln Ar #g Ala Ile Ala Pro Phe                805   #               810   #               815Thr Arg Gly Gly Asp Ile Ile Ala Gln Ala Gl #n Ser Gly Thr Gly Lys            820       #           825       #           830Thr Gly Ala Phe Ser Ile Gly Leu Leu Gln Ar #g Leu Asp Phe Arg His        835           #       840           #       845Asn Leu Ile Gln Gly Leu Val Leu Ser Pro Th #r Arg Glu Leu Ala Leu    850               #   855               #   860Gln Thr Ala Glu Val Ile Ser Arg Ile Gly Gl #u Phe Leu Ser Asn Ser865                 8 #70                 8 #75                 8 #80Ser Lys Phe Cys Glu Thr Phe Val Gly Gly Th #r Arg Val Gln Asp Asp                885   #               890   #               895Leu Arg Lys Leu Gln Ala Gly Val Ile Val Al #a Val Gly Thr Pro Gly            900       #           905       #           910Arg Val Ser Asp Val Ile Lys Arg Gly Ala Le #u Arg Thr Glu Ser Leu        915           #       920           #       925Arg Val Leu Val Leu Asp Glu Ala Asp Glu Me #t Leu Ser Gln Gly Phe    930               #   935               #   940Ala Asp Gln Ile Tyr Glu Ile Phe Arg Phe Le #u Pro Lys Asp Ile Gln945                 9 #50                 9 #55                 9 #60Val Ala Leu Phe Ser Ala Thr Met Pro Glu Gl #u Val Leu Glu Leu Thr                965   #               970   #               975Lys Lys Phe Met Arg Asp             980 <210> SEQ ID NO 96<211> LENGTH: 1641 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Fusion (poly-protein) constr#ucts comprising       multiple Leishmania antigens <400> SEQUENCE: 96Met His His His His His His Met Ser Cys Gl #y Asn Ala Lys Ile Asn                   #5                   #10                   #15Ser Pro Ala Pro Ser Phe Glu Glu Val Ala Le #u Met Pro Asn Gly Ser             20      #             25      #             30Phe Lys Lys Ile Ser Leu Ser Ser Tyr Lys Gl #y Lys Trp Val Val Leu         35          #         40          #         45Phe Phe Tyr Pro Leu Asp Phe Thr Phe Val Cy #s Pro Thr Glu Val Ile     50              #     55              #     60Ala Phe Ser Asp Ser Val Ser Arg Phe Asn Gl #u Leu Asn Cys Glu Val 65                  # 70                  # 75                  # 80Leu Ala Cys Ser Ile Asp Ser Glu Tyr Ala Hi #s Leu Gln Trp Thr Leu                 85  #                 90  #                 95Gln Asp Arg Lys Lys Gly Gly Leu Gly Thr Me #t Ala Ile Pro Met Leu            100       #           105       #           110Ala Asp Lys Thr Lys Ser Ile Ala Arg Ser Ty #r Gly Val Leu Glu Glu        115           #       120           #       125Ser Gln Gly Val Ala Tyr Arg Gly Leu Phe Il #e Ile Asp Pro His Gly    130               #   135               #   140Met Leu Arg Gln Ile Thr Val Asn Asp Met Pr #o Val Gly Arg Ser Val145                 1 #50                 1 #55                 1 #60Glu Glu Val Leu Arg Leu Leu Glu Ala Phe Gl #n Phe Val Glu Lys His                165   #               170   #               175Gly Glu Val Cys Pro Ala Asn Trp Lys Lys Gl #y Ala Pro Thr Met Lys            180       #           185       #           190Pro Glu Pro Asn Ala Ser Val Glu Gly Tyr Ph #e Ser Lys Gln Gly Ser        195           #       200           #       205Met Asp Ala Thr Glu Leu Lys Asn Lys Gly As #n Glu Glu Phe Ser Ala    210               #   215               #   220Gly Arg Tyr Val Glu Ala Val Asn Tyr Phe Se #r Lys Ala Ile Gln Leu225                 2 #30                 2 #35                 2 #40Asp Glu Gln Asn Ser Val Leu Tyr Ser Asn Ar #g Ser Ala Cys Phe Ala                245   #               250   #               255Ala Met Gln Lys Tyr Lys Asp Ala Leu Asp As #p Ala Asp Lys Cys Ile            260       #           265       #           270Ser Ile Lys Pro Asn Trp Ala Lys Gly Tyr Va #l Arg Arg Gly Ala Ala        275           #       280           #       285Leu His Gly Met Arg Arg Tyr Asp Asp Ala Il #e Ala Ala Tyr Glu Lys    290               #   295               #   300Gly Leu Lys Val Asp Pro Ser Asn Ser Gly Cy #s Ala Gln Gly Val Lys305                 3 #10                 3 #15                 3 #20Asp Val Gln Val Ala Lys Ala Arg Glu Ala Ar #g Asp Pro Ile Ala Arg                325   #               330   #               335Val Phe Thr Pro Glu Ala Phe Arg Lys Ile Gl #n Glu Asn Pro Lys Leu            340       #           345       #           350Ser Leu Leu Met Leu Gln Pro Asp Tyr Val Ly #s Met Val Asp Thr Val        355           #       360           #       365Ile Arg Asp Pro Ser Gln Gly Arg Leu Tyr Me #t Glu Asp Gln Arg Phe    370               #   375               #   380Ala Leu Thr Leu Met Tyr Leu Ser Gly Met Ly #s Ile Pro Asn Asp Gly385                 3 #90                 3 #95                 4 #00Asp Gly Glu Glu Glu Glu Arg Pro Ser Ala Ly #s Ala Ala Glu Thr Ala                405   #               410   #               415Lys Pro Lys Glu Glu Lys Pro Leu Thr Asp As #n Glu Lys Glu Ala Leu            420       #           425       #           430Ala Leu Lys Glu Glu Gly Asn Lys Leu Tyr Le #u Ser Lys Lys Phe Glu        435           #       440           #       445Glu Ala Leu Thr Lys Tyr Gln Glu Ala Gln Va #l Lys Asp Pro Asn Asn    450               #   455               #   460Thr Leu Tyr Ile Leu Asn Val Ser Ala Val Ty #r Phe Glu Gln Gly Asp465                 4 #70                 4 #75                 4 #80Tyr Asp Lys Cys Ile Ala Glu Cys Glu His Gl #y Ile Glu His Gly Arg                485   #               490   #               495Glu Asn His Cys Asp Tyr Thr Ile Ile Ala Ly #s Leu Met Thr Arg Asn            500       #           505       #           510Ala Leu Cys Leu Gln Arg Gln Arg Lys Tyr Gl #u Ala Ala Ile Asp Leu        515           #       520           #       525Tyr Lys Arg Ala Leu Val Glu Trp Arg Asn Pr #o Asp Thr Leu Lys Lys    530               #   535               #   540Leu Thr Glu Cys Glu Lys Glu His Gln Lys Al #a Val Glu Glu Ala Tyr545                 5 #50                 5 #55                 5 #60Ile Asp Pro Glu Ile Ala Lys Gln Lys Lys As #p Glu Gly Asn Gln Tyr                565   #               570   #               575Phe Lys Glu Asp Lys Phe Pro Glu Ala Val Al #a Ala Tyr Thr Glu Ala            580       #           585       #           590Ile Lys Arg Asn Pro Ala Glu His Thr Ser Ty #r Ser Asn Arg Ala Ala        595           #       600           #       605Ala Tyr Ile Lys Leu Gly Ala Phe Asn Asp Al #a Leu Lys Asp Ala Glu    610               #   615               #   620Lys Cys Ile Glu Leu Lys Pro Asp Phe Val Ly #s Gly Tyr Ala Arg Lys625                 6 #30                 6 #35                 6 #40Gly His Ala Tyr Phe Trp Thr Lys Gln Tyr As #n Arg Ala Leu Gln Ala                645   #               650   #               655Tyr Asn Glu Gly Leu Lys Val Asp Pro Ser As #n Ala Asp Cys Lys Asp            660       #           665       #           670Gly Arg Tyr Arg Thr Ile Met Lys Ile Gln Gl #u Met Ala Ser Gly Gln        675           #       680           #       685Ser Ala Asp Gly Asp Glu Ala Ala Arg Arg Al #a Met Asp Asp Pro Glu    690               #   695               #   700Ile Ala Ala Ile Met Gln Asp Ser Tyr Met Gl #n Leu Val Leu Lys Glu705                 7 #10                 7 #15                 7 #20Met Gln Asn Asp Pro Thr Arg Ile Gln Glu Ty #r Met Lys Asp Ser Gly                725   #               730   #               735Ile Ser Ser Lys Ile Asn Lys Leu Ile Ser Al #a Gly Ile Ile Arg Phe            740       #           745       #           750Gly Gln Glu Phe Ser Leu Thr Asp Pro Ala Va #l Leu Gly Glu Glu Thr        755           #       760           #       765His Leu Arg Val Arg Val Val Pro Asp Lys Al #a Asn Lys Thr Leu Thr    770               #   775               #   780Val Glu Asp Asn Gly Ile Gly Met Thr Lys Al #a Asp Leu Val Asn Asn785                 7 #90                 7 #95                 8 #00Leu Gly Thr Ile Ala Arg Ser Gly Thr Lys Al #a Phe Met Glu Ala Leu                805   #               810   #               815Glu Ala Gly Gly Asp Met Ser Met Ile Gly Gl #n Phe Gly Val Gly Phe            820       #           825       #           830Tyr Ser Ala Tyr Leu Val Ala Asp Arg Val Th #r Val Val Ser Lys Asn        835           #       840           #       845Asn Ser Asp Glu Ala Tyr Val Trp Glu Ser Se #r Ala Gly Gly Thr Phe    850               #   855               #   860Thr Ile Thr Ser Val Pro Glu Ser Asp Met Ly #s Arg Gly Thr Arg Ile865                 8 #70                 8 #75                 8 #80Thr Leu His Leu Lys Glu Asp Gln Gln Glu Ty #r Leu Glu Glu Arg Arg                885   #               890   #               895Val Lys Glu Leu Ile Lys Lys His Ser Glu Ph #e Ile Gly Tyr Asp Ile            900       #           905       #           910Glu Leu Met Val Glu Lys Thr Ala Glu Lys Gl #u Val Thr Asp Glu Asp        915           #       920           #       925Glu Glu Glu Asp Glu Ser Lys Lys Lys Ser Cy #s Gly Asp Glu Gly Glu    930               #   935               #   940Pro Lys Val Glu Glu Val Thr Glu Gly Gly Gl #u Asp Lys Lys Lys Lys945                 9 #50                 9 #55                 9 #60Thr Lys Lys Val Lys Glu Val Thr Lys Thr Ty #r Glu Val Gln Asn Lys                965   #               970   #               975His Lys Pro Leu Trp Thr Arg Asp Pro Lys As #p Val Thr Lys Glu Glu            980       #           985       #           990Tyr Ala Ala Phe Tyr Lys Ala Ile Ser Asn As #p Trp Glu Asp Pro Ala        995           #       1000           #      1005Ala Thr Lys His Phe Ser Val Glu Gly Gln Le #u Glu Phe Arg Ala Ile    1010              #   1015               #  1020Ala Phe Val Pro Lys Arg Ala Pro Phe Asp Me #t Phe Glu Pro Asn Lys1025                1030 #                1035  #               1040Lys Arg Asn Asn Ile Lys Leu Tyr Val Arg Ar #g Val Phe Ile Met Asp                1045  #               1050   #              1055Asn Cys Glu Asp Leu Cys Pro Asp Trp Leu Gl #y Phe Val Lys Gly Val            1060      #           1065       #          1070Val Asp Ser Glu Asp Leu Pro Leu Asn Ile Se #r Arg Glu Asn Leu Gln        1075          #       1080           #      1085Gln Asn Lys Ile Leu Lys Val Ile Arg Lys As #n Ile Val Lys Lys Cys    1090              #   1095               #  1100Leu Glu Leu Phe Glu Glu Ile Ala Glu Asn Ly #s Glu Asp Tyr Lys Gln1105                1110 #                1115  #               1120Phe Tyr Glu Gln Phe Gly Lys Asn Ile Lys Le #u Gly Ile His Glu Asp                1125  #               1130   #              1135Thr Ala Asn Arg Lys Lys Leu Met Glu Leu Le #u Arg Phe Tyr Ser Thr            1140      #           1145       #          1150Glu Ser Gly Glu Glu Met Thr Thr Leu Lys As #p Tyr Val Thr Arg Met        1155          #       1160           #      1165Lys Pro Glu Gln Lys Ser Ile Tyr Tyr Ile Th #r Gly Asp Ser Lys Lys    1170              #   1175               #  1180Lys Leu Glu Ser Ser Pro Phe Ile Glu Lys Al #a Arg Arg Cys Gly Leu1185                1190 #                1195  #               1200Glu Val Leu Phe Met Thr Glu Pro Ile Asp Gl #u Tyr Val Met Gln Gln                1205  #               1210   #              1215Val Lys Asp Phe Glu Asp Lys Lys Phe Ala Cy #s Leu Thr Lys Glu Gly            1220      #           1225       #          1230Val His Phe Glu Glu Ser Glu Glu Glu Lys Ly #s Gln Arg Glu Glu Lys        1235          #       1240           #      1245Lys Ala Ala Cys Glu Lys Leu Cys Lys Thr Me #t Lys Glu Val Leu Gly    1250              #   1255               #  1260Asp Lys Val Glu Lys Val Thr Val Ser Glu Ar #g Leu Ser Thr Ser Pro1265                1270 #                1275  #               1280Cys Ile Leu Val Thr Ser Glu Phe Gly Trp Se #r Ala His Met Glu Gln                1285  #               1290   #              1295Ile Met Arg Asn Gln Ala Leu Arg Asp Ser Se #r Met Ala Gln Tyr Met            1300      #           1305       #          1310Val Ser Lys Lys Thr Met Glu Val Asn Pro As #p His Pro Ile Ile Lys        1315          #       1320           #      1325Glu Leu Arg Arg Arg Val Glu Ala Asp Glu As #n Asp Lys Ala Val Lys    1330              #   1335               #  1340Asp Leu Val Phe Leu Leu Phe Asp Thr Ser Le #u Leu Thr Ser Gly Phe1345                1350 #                1355  #               1360Gln Leu Asp Asp Pro Thr Gly Tyr Ala Glu Ar #g Ile Asn Arg Met Ile                1365  #               1370   #              1375Lys Leu Gly Leu Ser Leu Asp Glu Glu Glu Gl #u Glu Val Ala Glu Ala            1380      #           1385       #          1390Pro Pro Ala Glu Ala Ala Pro Ala Glu Val Th #r Ala Gly Thr Ser Ser        1395          #       1400           #      1405Met Glu Gln Val Asp Asp Ile Met Ala Gln As #n Asp Lys Ile Ala Pro    1410              #   1415               #  1420Gln Asp Gln Asp Ser Phe Leu Asp Asp Gln Pr #o Gly Val Arg Pro Ile1425                1430 #                1435  #               1440Pro Ser Phe Asp Asp Met Pro Leu His Gln As #n Leu Leu Arg Gly Ile                1445  #               1450   #              1455Tyr Ser Tyr Gly Phe Glu Lys Pro Ser Ser Il #e Gln Gln Arg Ala Ile            1460      #           1465       #          1470Ala Pro Phe Thr Arg Gly Gly Asp Ile Ile Al #a Gln Ala Gln Ser Gly        1475          #       1480           #      1485Thr Gly Lys Thr Gly Ala Phe Ser Ile Gly Le #u Leu Gln Arg Leu Asp    1490              #   1495               #  1500Phe Arg His Asn Leu Ile Gln Gly Leu Val Le #u Ser Pro Thr Arg Glu1505                1510 #                1515  #               1520Leu Ala Leu Gln Thr Ala Glu Val Ile Ser Ar #g Ile Gly Glu Phe Leu                1525  #               1530   #              1535Ser Asn Ser Ser Lys Phe Cys Glu Thr Phe Va #l Gly Gly Thr Arg Val            1540      #           1545       #          1550Gln Asp Asp Leu Arg Lys Leu Gln Ala Gly Va #l Ile Val Ala Val Gly        1555          #       1560           #      1565Thr Pro Gly Arg Val Ser Asp Val Ile Lys Ar #g Gly Ala Leu Arg Thr    1570              #   1575               #  1580Glu Ser Leu Arg Val Leu Val Leu Asp Glu Al #a Asp Glu Met Leu Ser1585                1590 #                1595  #               1600Gln Gly Phe Ala Asp Gln Ile Tyr Glu Ile Ph #e Arg Phe Leu Pro Lys                1605  #               1610   #              1615Asp Ile Gln Val Ala Leu Phe Ser Ala Thr Me #t Pro Glu Glu Val Leu            1620      #           1625       #          1630Glu Leu Thr Lys Lys Phe Met Arg Asp         1635          #       1640<210> SEQ ID NO 97 <211> LENGTH: 1427 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Fusion (poly-protein) constr #ucts comprising      multiple Leishmania antigens <400> SEQUENCE: 97Met His His His His His His Met Ser Cys Gl #y Asn Ala Lys Ile Asn                   #5                   #10                   #15Ser Pro Ala Pro Ser Phe Glu Glu Val Ala Le #u Met Pro Asn Gly Ser             20      #             25      #             30Phe Lys Lys Ile Ser Leu Ser Ser Tyr Lys Gl #y Lys Trp Val Val Leu         35          #         40          #         45Phe Phe Tyr Pro Leu Asp Phe Thr Phe Val Cy #s Pro Thr Glu Val Ile     50              #     55              #     60Ala Phe Ser Asp Ser Val Ser Arg Phe Asn Gl #u Leu Asn Cys Glu Val 65                  # 70                  # 75                  # 80Leu Ala Cys Ser Ile Asp Ser Glu Tyr Ala Hi #s Leu Gln Trp Thr Leu                 85  #                 90  #                 95Gln Asp Arg Lys Lys Gly Gly Leu Gly Thr Me #t Ala Ile Pro Met Leu            100       #           105       #           110Ala Asp Lys Thr Lys Ser Ile Ala Arg Ser Ty #r Gly Val Leu Glu Glu        115           #       120           #       125Ser Gln Gly Val Ala Tyr Arg Gly Leu Phe Il #e Ile Asp Pro His Gly    130               #   135               #   140Met Leu Arg Gln Ile Thr Val Asn Asp Met Pr #o Val Gly Arg Ser Val145                 1 #50                 1 #55                 1 #60Glu Glu Val Leu Arg Leu Leu Glu Ala Phe Gl #n Phe Val Glu Lys His                165   #               170   #               175Gly Glu Val Cys Pro Ala Asn Trp Lys Lys Gl #y Ala Pro Thr Met Lys            180       #           185       #           190Pro Glu Pro Asn Ala Ser Val Glu Gly Tyr Ph #e Ser Lys Gln Gly Ser        195           #       200           #       205Met Asp Ala Thr Glu Leu Lys Asn Lys Gly As #n Glu Glu Phe Ser Ala    210               #   215               #   220Gly Arg Tyr Val Glu Ala Val Asn Tyr Phe Se #r Lys Ala Ile Gln Leu225                 2 #30                 2 #35                 2 #40Asp Glu Gln Asn Ser Val Leu Tyr Ser Asn Ar #g Ser Ala Cys Phe Ala                245   #               250   #               255Ala Met Gln Lys Tyr Lys Asp Ala Leu Asp As #p Ala Asp Lys Cys Ile            260       #           265       #           270Ser Ile Lys Pro Asn Trp Ala Lys Gly Tyr Va #l Arg Arg Gly Ala Ala        275           #       280           #       285Leu His Gly Met Arg Arg Tyr Asp Asp Ala Il #e Ala Ala Tyr Glu Lys    290               #   295               #   300Gly Leu Lys Val Asp Pro Ser Asn Ser Gly Cy #s Ala Gln Gly Val Lys305                 3 #10                 3 #15                 3 #20Asp Val Gln Val Ala Lys Ala Arg Glu Ala Ar #g Asp Pro Ile Ala Arg                325   #               330   #               335Val Phe Thr Pro Glu Ala Phe Arg Lys Ile Gl #n Glu Asn Pro Lys Leu            340       #           345       #           350Ser Leu Leu Met Leu Gln Pro Asp Tyr Val Ly #s Met Val Asp Thr Val        355           #       360           #       365Ile Arg Asp Pro Ser Gln Gly Arg Leu Tyr Me #t Glu Asp Gln Arg Phe    370               #   375               #   380Ala Leu Thr Leu Met Tyr Leu Ser Gly Met Ly #s Ile Pro Asn Asp Gly385                 3 #90                 3 #95                 4 #00Asp Gly Glu Glu Glu Glu Arg Pro Ser Ala Ly #s Ala Ala Glu Thr Ala                405   #               410   #               415Lys Pro Lys Glu Glu Lys Pro Leu Thr Asp As #n Glu Lys Glu Ala Leu            420       #           425       #           430Ala Leu Lys Glu Glu Gly Asn Lys Leu Tyr Le #u Ser Lys Lys Phe Glu        435           #       440           #       445Glu Ala Leu Thr Lys Tyr Gln Glu Ala Gln Va #l Lys Asp Pro Asn Asn    450               #   455               #   460Thr Leu Tyr Ile Leu Asn Val Ser Ala Val Ty #r Phe Glu Gln Gly Asp465                 4 #70                 4 #75                 4 #80Tyr Asp Lys Cys Ile Ala Glu Cys Glu His Gl #y Ile Glu His Gly Arg                485   #               490   #               495Glu Asn His Cys Asp Tyr Thr Ile Ile Ala Ly #s Leu Met Thr Arg Asn            500       #           505       #           510Ala Leu Cys Leu Gln Arg Gln Arg Lys Tyr Gl #u Ala Ala Ile Asp Leu        515           #       520           #       525Tyr Lys Arg Ala Leu Val Glu Trp Arg Asn Pr #o Asp Thr Leu Lys Lys    530               #   535               #   540Leu Thr Glu Cys Glu Lys Glu His Gln Lys Al #a Val Glu Glu Ala Tyr545                 5 #50                 5 #55                 5 #60Ile Asp Pro Glu Ile Ala Lys Gln Lys Lys As #p Glu Gly Asn Gln Tyr                565   #               570   #               575Phe Lys Glu Asp Lys Phe Pro Glu Ala Val Al #a Ala Tyr Thr Glu Ala            580       #           585       #           590Ile Lys Arg Asn Pro Ala Glu His Thr Ser Ty #r Ser Asn Arg Ala Ala        595           #       600           #       605Ala Tyr Ile Lys Leu Gly Ala Phe Asn Asp Al #a Leu Lys Asp Ala Glu    610               #   615               #   620Lys Cys Ile Glu Leu Lys Pro Asp Phe Val Ly #s Gly Tyr Ala Arg Lys625                 6 #30                 6 #35                 6 #40Gly His Ala Tyr Phe Trp Thr Lys Gln Tyr As #n Arg Ala Leu Gln Ala                645   #               650   #               655Tyr Asn Glu Gly Leu Lys Val Asp Pro Ser As #n Ala Asp Cys Lys Asp            660       #           665       #           670Gly Arg Tyr Arg Thr Ile Met Lys Ile Gln Gl #u Met Ala Ser Gly Gln        675           #       680           #       685Ser Ala Asp Gly Asp Glu Ala Ala Arg Arg Al #a Met Asp Asp Pro Glu    690               #   695               #   700Ile Ala Ala Ile Met Gln Asp Ser Tyr Met Gl #n Leu Val Leu Lys Glu705                 7 #10                 7 #15                 7 #20Met Gln Asn Asp Pro Thr Arg Ile Gln Glu Ty #r Met Lys Asp Ser Gly                725   #               730   #               735Ile Ser Ser Lys Ile Asn Lys Leu Ile Ser Al #a Gly Ile Ile Arg Phe            740       #           745       #           750Gly Gln Glu Phe Ser Leu Thr Asp Pro Ala Va #l Leu Gly Glu Glu Thr        755           #       760           #       765His Leu Arg Val Arg Val Val Pro Asp Lys Al #a Asn Lys Thr Leu Thr    770               #   775               #   780Val Glu Asp Asn Gly Ile Gly Met Thr Lys Al #a Asp Leu Val Asn Asn785                 7 #90                 7 #95                 8 #00Leu Gly Thr Ile Ala Arg Ser Gly Thr Lys Al #a Phe Met Glu Ala Leu                805   #               810   #               815Glu Ala Gly Gly Asp Met Ser Met Ile Gly Gl #n Phe Gly Val Gly Phe            820       #           825       #           830Tyr Ser Ala Tyr Leu Val Ala Asp Arg Val Th #r Val Val Ser Lys Asn        835           #       840           #       845Asn Ser Asp Glu Ala Tyr Val Trp Glu Ser Se #r Ala Gly Gly Thr Phe    850               #   855               #   860Thr Ile Thr Ser Val Pro Glu Ser Asp Met Ly #s Arg Gly Thr Arg Ile865                 8 #70                 8 #75                 8 #80Thr Leu His Leu Lys Glu Asp Gln Gln Glu Ty #r Leu Glu Glu Arg Arg                885   #               890   #               895Val Lys Glu Leu Ile Lys Lys His Ser Glu Ph #e Ile Gly Tyr Asp Ile            900       #           905       #           910Glu Leu Met Val Glu Lys Thr Ala Glu Lys Gl #u Val Thr Asp Glu Asp        915           #       920           #       925Glu Glu Glu Asp Glu Ser Lys Lys Lys Ser Cy #s Gly Asp Glu Gly Glu    930               #   935               #   940Pro Lys Val Glu Glu Val Thr Glu Gly Gly Gl #u Asp Lys Lys Lys Lys945                 9 #50                 9 #55                 9 #60Thr Lys Lys Val Lys Glu Val Thr Lys Thr Ty #r Glu Val Gln Asn Lys                965   #               970   #               975His Lys Pro Leu Trp Thr Arg Asp Pro Lys As #p Val Thr Lys Glu Glu            980       #           985       #           990Tyr Ala Ala Phe Tyr Lys Ala Ile Ser Asn As #p Trp Glu Asp Pro Ala        995           #       1000           #      1005Ala Thr Lys His Phe Ser Val Glu Gly Gln Le #u Glu Phe Arg Ala Ile    1010              #   1015               #  1020Ala Phe Val Pro Lys Arg Ala Pro Phe Asp Me #t Phe Glu Pro Asn Lys1025                1030 #                1035  #               1040Lys Arg Asn Asn Ile Lys Leu Tyr Val Arg Ar #g Val Phe Ile Met Asp                1045  #               1050   #              1055Asn Cys Glu Asp Leu Cys Pro Asp Trp Leu Gl #y Phe Val Lys Gly Val            1060      #           1065       #          1070Val Asp Ser Glu Asp Leu Pro Leu Asn Ile Se #r Arg Glu Asn Leu Gln        1075          #       1080           #     1085Gln Asn Lys Ile Leu Lys Val Ile Arg Lys As #n Ile Val Lys Lys Cys    1090              #   1095               #  1100Leu Glu Leu Phe Glu Glu Ile Ala Glu Asn Ly #s Glu Asp Tyr Lys Gln1105                1110 #                1115  #               1120Phe Tyr Glu Gln Phe Gly Lys Asn Ile Lys Le #u Gly Ile His Glu Asp                1125  #               1130   #              1135Thr Ala Asn Arg Lys Lys Leu Met Glu Leu Le #u Arg Phe Tyr Ser Thr            1140      #           1145       #          1150Glu Ser Gly Glu Glu Met Thr Thr Leu Lys As #p Tyr Val Thr Arg Met        1155          #       1160           #      1165Lys Pro Glu Gln Lys Ser Ile Tyr Tyr Ile Th #r Gly Asp Ser Lys Lys    1170              #   1175               #  1180Lys Leu Glu Ser Ser Pro Phe Ile Glu Lys Al #a Arg Arg Cys Gly Leu1185                1190 #                1195  #               1200Glu Val Leu Phe Met Thr Glu Pro Ile Asp Gl #u Tyr Val Met Gln Gln                1205  #               1210   #              1215Val Lys Asp Phe Glu Asp Lys Lys Phe Ala Cy #s Leu Thr Lys Glu Gly            1220      #           1225       #          1230Val His Phe Glu Glu Ser Glu Glu Glu Lys Ly #s Gln Arg Glu Glu Lys        1235          #       1240           #      1245Lys Ala Ala Cys Glu Lys Leu Cys Lys Thr Me #t Lys Glu Val Leu Gly    1250              #   1255               #  1260Asp Lys Val Glu Lys Val Thr Val Ser Glu Ar #g Leu Ser Thr Ser Pro1265                1270 #                1275  #               1280Cys Ile Leu Val Thr Ser Glu Phe Gly Trp Se #r Ala His Met Glu Gln                1285  #               1290   #              1295Ile Met Arg Asn Gln Ala Leu Arg Asp Ser Se #r Met Ala Gln Tyr Met            1300      #           1305       #          1310Val Ser Lys Lys Thr Met Glu Val Asn Pro As #p His Pro Ile Ile Lys        1315          #       1320           #      1325Glu Leu Arg Arg Arg Val Glu Ala Asp Glu As #n Asp Lys Ala Val Lys    1330              #   1335               #  1340Asp Leu Val Phe Leu Leu Phe Asp Thr Ser Le #u Leu Thr Ser Gly Phe1345                1350 #                1355  #               1360Gln Leu Asp Asp Pro Thr Gly Tyr Ala Glu Ar #g Ile Asn Arg Met Ile                1365  #               1370   #              1375Lys Leu Gly Leu Ser Leu Asp Glu Glu Glu Gl #u Glu Val Ala Glu Ala            1380      #           1385       #          1390Pro Pro Ala Glu Ala Ala Pro Ala Glu Val Th #r Ala Gly Thr Ser Ser        1395          #       1400           #      1405Met Glu Gln Val Asp Asp Ile His His Thr Gl #y Gly Arg Ser Ser Arg    1410              #   1415               #  1420 Ser Gly Cys 1425<210> SEQ ID NO 98 <211> LENGTH: 4929 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: DNA sequence encoding fus #ion (poly-protein)      constructs comprising multiple Leishman #ia antigens<400> SEQUENCE: 98catatgcacc accaccacca ccacatgtcc tgcggtaacg ccaagatcaa ct#ctcccgcg     60ccgtccttcg aggaggtggc gctcatgccc aacggcagct tcaagaagat ca#gcctctcc    120tcctacaagg gcaagtgggt cgtgctcttc ttctacccgc tcgacttcac ct#tcgtgtgc    180ccgacagagg tcatcgcgtt ctccgacagc gtgagtcgct tcaacgagct ca#actgcgag    240gtcctcgcgt gctcgataga cagcgagtac gcgcacctgc agtggacgct gc#aggaccgc    300aagaagggcg gcctcgggac catggcgatc ccaatgctag ccgacaagac ca#agagcatc    360gctcgttcct acggcgtgct ggaggagagc cagggcgtgg cctaccgcgg tc#tcttcatc    420atcgaccccc atggcatgct gcgtcagatc accgtcaatg acatgccggt gg#gccgcagc    480gtggaggagg ttctacgcct gctggaggct tttcagttcg tggagaagca cg#gcgaggtg    540tgccccgcga actggaagaa gggcgccccc acgatgaagc cggaaccgaa tg#cgtctgtc    600gagggatact tcagcaagca gggatccatg gacgcaactg agctgaagaa ca#aggggaac    660gaagagttct ccgccggccg ctatgtggag gcggtgaact acttctcaaa gg#cgatccag    720ttggatgagc agaacagtgt cctctacagc aaccgctccg cctgttttgc ag#ccatgcag    780aaatacaagg acgcgctgga cgacgccgac aagtgcatct cgatcaagcc ga#attgggcc    840aagggctacg tgcgccgagg agcagctctc catggcatgc gccgctacga cg#atgccatt    900gccgcgtatg aaaaggggct caaggtggac ccttccaaca gcggctgcgc gc#agggcgtg    960aaggacgtgc aggtagccaa ggcccgcgaa gcacgtgacc ccatcgctcg cg#tcttcacc   1020ccggaggcgt tccgcaagat ccaagagaat cccaagctgt ctctacttat gc#tgcagccg   1080gactacgtga agatggtaga caccgtcatc cgcgaccctt cgcagggccg gc#tgtacatg   1140gaagaccagc gctttgccct gacgctcatg tacctgagcg gaatgaagat tc#ccaacgat   1200ggtgatggcg aggaggagga acgtccgtct gcgaaggcgg cagagacagc ga#agccaaaa   1260gaggagaagc ctctcaccga caacgagaag gaggccctgg cgctcaagga gg#agggcaac   1320aagctgtacc tctcgaagaa gtttgaggag gcgctgacca agtaccaaga gg#cgcaggtg   1380aaagacccca acaacacttt atacattctg aacgtgtcgg ccgtgtactt cg#agcagggt   1440gactacgaca agtgcatcgc cgagtgcgag cacggtatcg agcacggtcg cg#agaaccac   1500tgcgactaca caatcattgc gaagctcatg acccggaacg ccttgtgcct cc#agaggcag   1560aggaagtacg aggctgctat cgacctttac aagcgcgccc ttgtcgagtg gc#gtaaccct   1620gacaccctca agaagctgac ggagtgcgag aaggagcacc aaaaggcggt gg#aggaagcc   1680tacatcgatc ctgagatcgc gaagcagaag aaagacgaag gtaaccagta ct#tcaaggag   1740gataagttcc ccgaggccgt ggcagcgtac acggaggcca tcaagcgcaa cc#ctgccgag   1800cacacctcct acagcaatcg cgcggccgcg tacatcaagc ttggagcctt ca#acgacgcc   1860ctcaaggacg cggagaagtg cattgagctg aagcccgact ttgttaaggg ct#acgcgcgc   1920aagggtcatg cttacttttg gaccaagcag tacaaccgcg cgctgcaggc gt#acaatgag   1980ggcctcaagg tggacccgag caatgcggac tgcaaggatg ggcggtatcg ca#caatcatg   2040aagattcagg agatggcatc tggccaatcc gcggatggcg acgaggcggc gc#gccgggcc   2100atggacgatc ctgaaatcgc ggcaatcatg caagatagct acatgcaact ag#tgttgaag   2160gagatgcaga acgatcccac gcgcattcag gagtacatga aggactccgg ga#tctcatcg   2220aagatcaaca agctgatttc agctggcatc attcgttttg gtcaggaatt ca#gcctgacg   2280gacccggcgg tgctgggcga ggagactcac ctgcgcgtcc gcgtggtgcc gg#acaaggcg   2340aacaagacgc tgacggtgga ggataacggc atcggcatga ccaaggcgga cc#tcgtgaac   2400aatctgggca cgatcgcgcg ctccggcacg aaggcgttca tggaggcact gg#aggccggc   2460ggcgacatga gcatgatcgg ccagttcggt gtcggcttct actccgcgta cc#ttgtggcg   2520gaccgcgtga cggtggtgtc gaagaacaac tcggacgagg cgtacgtatg gg#agtcgtcc   2580gcgggcggca cgttcaccat cacgagcgtg ccggagtcgg acatgaagcg cg#gcacgcgc   2640atcacgctgc acctaaagga ggaccagcag gagtacctgg aggagcgccg gg#tgaaggag   2700ctgatcaaga agcactccga gttcatcggc tacgacatcg agctgatggt gg#agaagacg   2760gcggagaagg aggtgacgga cgaggacgag gaggaggacg agtcgaagaa ga#agtcctgc   2820ggggacgagg gcgagccgaa ggtggaggag gtgacggagg gcggcgagga ca#agaagaag   2880aagacgaaga aggtgaagga ggtgacgaag acgtacgagg tccagaacaa gc#acaagccg   2940ctctggacgc gcgacccgaa ggacgtgacg aaggaggagt acgcggcctt ct#acaaggcc   3000atctccaacg actgggagga cccggcggcg acgaagcact tctcggtgga gg#gccagctg   3060gagttccgcg cgatcgcgtt cgtgccgaag cgcgcgccgt tcgacatgtt cg#agccgaac   3120aagaagcgca acaacatcaa gctgtacgtg cgccgcgtgt tcatcatgga ca#actgcgag   3180gacctgtgcc cggactggct cggcttcgtg aagggcgtcg tggacagcga gg#acctgccg   3240ctgaacatct cgcgcgagaa cctgcagcag aacaagatcc tgaaggtgat cc#gcaagaac   3300atcgtgaaga agtgcctgga gctgttcgaa gagatagcgg agaacaagga gg#actacaag   3360cagttctacg agcagttcgg caagaacatc aagctgggca tccacgagga ca#cggcgaac   3420cgcaagaagc tgatggagtt gctgcgcttc tacagcaccg agtcggggga gg#agatgacg   3480acactgaagg actacgtgac gcgcatgaag ccggagcaga agtcgatcta ct#acatcact   3540ggcgacagca agaagaagct ggagtcgtcg ccgttcatcg agaaggcgag ac#gctgcggg   3600ctcgaggtgc tgttcatgac ggagccgatc gacgagtacg tgatgcagca gg#tgaaggac   3660ttcgaggaca agaagttcgc gtgcctgacg aaggaaggcg tgcacttcga gg#agtccgag   3720gaggagaaga agcagcgcga ggagaagaag gcggcgtgcg agaagctgtg ca#agacgatg   3780aaggaggtgc tgggcgacaa ggtggagaag gtgaccgtgt cggagcgcct gt#cgacgtcg   3840ccgtgcatcc tggtgacgtc ggagtttggg tggtcggcgc acatggaaca ga#tcatgcgc   3900aaccaggcgc tgcgcgactc cagcatggcg cagtacatgg tgtccaagaa ga#cgatggag   3960gtgaaccccg accaccccat catcaaggag ctgcgccgcc gcgtggaggc gg#acgagaac   4020gacaaggccg tgaaggacct cgtcttcctg ctcttcgaca cgtcgctgct ca#cgtccggc   4080ttccagctgg atgaccccac cggctacgcc gagcgcatca accgcatgat ca#agctcggc   4140ctgtcgctcg acgaggagga ggaggaggtc gccgaggcgc cgccggccga gg#cagccccc   4200gcggaggtca ccgccggcac ctccagcatg gagcaggtgg acgatatcat gg#cgcagaat   4260gataagatcg ccccccagga ccaggactcc ttcctcgatg accagcccgg cg#ttcgcccg   4320atcccgtcct tcgacgacat gccgctgcac cagaacctgc tgcgtggcat ct#actcgtac   4380gggttcgaga agccgtccag catccagcag cgcgcgatag cccccttcac gc#gcggcggc   4440gacatcatcg cgcaggccca gtccggtacc ggcaagacgg gtgccttctc ca#tcggtctg   4500ctgcagcgcc tggacttccg ccacaacctg atccagggcc tcgtgctctc cc#ccactcgc   4560gagctggccc tgcagacggc ggaggtgatc agccgcatcg gtgagttcct gt#cgaacagc   4620tccaagttct gcgagacctt tgtcggcggc acgcgcgtgc aggatgacct gc#gcaagctg   4680caggccggcg tcatcgttgc cgtgggcacg ccgggccgcg tgtccgacgt ga#tcaagcgt   4740ggcgcgctgc gcacagagtc gctgcgcgtg ctggtgctcg acgaggctga tg#agatgctg   4800tctcagggct tcgcggacca gatttacgag atcttccgct tcctgccgaa gg#acatccag   4860gtcgcgctct tctccgccac gatgccggag gaggtactgg agctgacgaa ga#agttcatg   4920 cgcgactaa                 #                  #                   #       4929 <210> SEQ ID NO 99 <211> LENGTH: 4233<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: DNA sequence encoding fus #ion (poly-protein)      constructs comprising multiple Leishman #ia antigens<400> SEQUENCE: 99ccagtgtggt ggatgtcctg cggtaacgcc aagatcaact ctcccgcgcc gt#ccttcgag     60gaggtggcgc tcatgcccaa cggcagcttc aagaagatca gcctctcctc ct#acaagggc    120aagtgggtcg tgctcttctt ctacccgctc gacttcacct tcgtgtgccc ga#cagaggtc    180atcgcgttct ccgacagcgt gagtcgcttc aacgagctca actgcgaggt cc#tcgcgtgc    240tcgatagaca gcgagtacgc gcacctgcag tggacgctgc aggaccgcaa ga#agggcggc    300ctcgggacca tggcgatccc aatgctagcc gacaagacca agagcatcgc tc#gttcctac    360ggcgtgctgg aggagagcca gggcgtggcc taccgcggtc tcttcatcat cg#acccccat    420ggcatgctgc gtcagatcac cgtcaatgac atgccggtgg gccgcagcgt gg#aggaggtt    480ctacgcctgc tggaggcttt tcagttcgtg gagaagcacg gcgaggtgtg cc#ccgcgaac    540tggaagaagg gcgcccccac gatgaagccg gaaccgaatg cgtctgtcga gg#gatacttc    600agcaagcagg gatccatgga cgcaactgag ctgaagaaca aggggaacga ag#agttctcc    660gccggccgct atgtggaggc ggtgaactac ttctcaaagg cgatccagtt gg#atgagcag    720aacagtgtcc tctacagcaa ccgctccgcc tgttttgcag ccatgcagaa at#acaaggac    780gcgctggacg acgccgacaa gtgcatctcg atcaagccga attgggccaa gg#gctacgtg    840cgccgaggag cagctctcca tggcatgcgc cgctacgacg atgccattgc cg#cgtatgaa    900aaggggctca aggtggaccc ttccaacagc ggctgcgcgc agggcgtgaa gg#acgtgcag    960gtagccaagg cccgcgaagc acgtgacccc atcgctcgcg tcttcacccc gg#aggcgttc   1020cgcaagatcc aagagaatcc caagctgtct ctacttatgc tgcagccgga ct#acgtgaag   1080atggtagaca ccgtcatccg cgacccttcg cagggccggc tgtacatgga ag#accagcgc   1140tttgccctga cgctcatgta cctgagcgga atgaagattc ccaacgatgg tg#atggcgag   1200gaggaggaac gtccgtctgc gaaggcggca gagacagcga agccaaaaga gg#agaagcct   1260ctcaccgaca acgagaagga ggccctggcg ctcaaggagg agggcaacaa gc#tgtacctc   1320tcgaagaagt ttgaggaggc gctgaccaag taccaagagg cgcaggtgaa ag#accccaac   1380aacactttat acattctgaa cgtgtcggcc gtgtacttcg agcagggtga ct#acgacaag   1440tgcatcgccg agtgcgagca cggtatcgag cacggtcgcg agaaccactg cg#actacaca   1500atcattgcga agctcatgac ccggaacgcc ttgtgcctcc agaggcagag ga#agtacgag   1560gctgctatcg acctttacaa gcgcgccctt gtcgagtggc gtaaccctga ca#ccctcaag   1620aagctgacgg agtgcgagaa ggagcaccaa aaggcggtgg aggaagccta ca#tcgatcct   1680gagatcgcga agcagaagaa agacgaaggt aaccagtact tcaaggagga ta#agttcccc   1740gaggccgtgg cagcgtacac ggaggccatc aagcgcaacc ctgccgagca ca#cctcctac   1800agcaatcgcg cggccgcgta catcaagctt ggagccttca acgacgccct ca#aggacgcg   1860gagaagtgca ttgagctgaa gcccgacttt gttaagggct acgcgcgcaa gg#gtcatgct   1920tacttttgga ccaagcagta caaccgcgcg ctgcaggcgt acaatgaggg cc#tcaaggtg   1980gacccgagca atgcggactg caaggatggg cggtatcgca caatcatgaa ga#ttcaggag   2040atggcatctg gccaatccgc ggatggcgac gaggcggcgc gccgggccat gg#acgatcct   2100gaaatcgcgg caatcatgca agatagctac atgcaactag tgttgaagga ga#tgcagaac   2160gatcccacgc gcattcagga gtacatgaag gactccggga tctcatcgaa ga#tcaacaag   2220ctgatttcag ctggcatcat tcgttttggt caggaattca gcctgacgga cc#cggcggtg   2280ctgggcgagg agactcacct gcgcgtccgc gtggtgccgg acaaggcgaa ca#agacgctg   2340acggtggagg ataacggcat cggcatgacc aaggcggacc tcgtgaacaa tc#tgggcacg   2400atcgcgcgct ccggcacgaa ggcgttcatg gaggcactgg aggccggcgg cg#acatgagc   2460atgatcggcc agttcggtgt cggcttctac tccgcgtacc ttgtggcgga cc#gcgtgacg   2520gtggtgtcga agaacaactc ggacgaggcg tacgtatggg agtcgtccgc gg#gcggcacg   2580ttcaccatca cgagcgtgcc ggagtcggac atgaagcgcg gcacgcgcat ca#cgctgcac   2640ctaaaggagg accagcagga gtacctggag gagcgccggg tgaaggagct ga#tcaagaag   2700cactccgagt tcatcggcta cgacatcgag ctgatggtgg agaagacggc gg#agaaggag   2760gtgacggacg aggacgagga ggaggacgag tcgaagaaga agtcctgcgg gg#acgagggc   2820gagccgaagg tggaggaggt gacggagggc ggcgaggaca agaagaagaa ga#cgaagaag   2880gtgaaggagg tgacgaagac gtacgaggtc cagaacaagc acaagccgct ct#ggacgcgc   2940gacccgaagg acgtgacgaa ggaggagtac gcggccttct acaaggccat ct#ccaacgac   3000tgggaggacc cggcggcgac gaagcacttc tcggtggagg gccagctgga gt#tccgcgcg   3060atcgcgttcg tgccgaagcg cgcgccgttc gacatgttcg agccgaacaa ga#agcgcaac   3120aacatcaagc tgtacgtgcg ccgcgtgttc atcatggaca actgcgagga cc#tgtgcccg   3180gactggctcg gcttcgtgaa gggcgtcgtg gacagcgagg acctgccgct ga#acatctcg   3240cgcgagaacc tgcagcagaa caagatcctg aaggtgatcc gcaagaacat cg#tgaagaag   3300tgcctggagc tgttcgaaga gatagcggag aacaaggagg actacaagca gt#tctacgag   3360cagttcggca agaacatcaa gctgggcatc cacgaggaca cggcgaaccg ca#agaagctg   3420atggagttgc tgcgcttcta cagcaccgag tcgggggagg agatgacgac ac#tgaaggac   3480tacgtgacgc gcatgaagcc ggagcagaag tcgatctact acatcactgg cg#acagcaag   3540aagaagctgg agtcgtcgcc gttcatcgag aaggcgagac gctgcgggct cg#aggtgctg   3600ttcatgacgg agccgatcga cgagtacgtg atgcagcagg tgaaggactt cg#aggacaag   3660aagttcgcgt gcctgacgaa ggaaggcgtg cacttcgagg agtccgagga gg#agaagaag   3720cagcgcgagg agaagaaggc ggcgtgcgag aagctgtgca agacgatgaa gg#aggtgctg   3780ggcgacaagg tggagaaggt gaccgtgtcg gagcgcctgt cgacgtcgcc gt#gcatcctg   3840gtgacgtcgg agtttgggtg gtcggcgcac atggaacaga tcatgcgcaa cc#aggcgctg   3900cgcgactcca gcatggcgca gtacatggtg tccaagaaga cgatggaggt ga#accccgac   3960caccccatca tcaaggagct gcgccgccgc gtggaggcgg acgagaacga ca#aggccgtg   4020aaggacctcg tcttcctgct cttcgacacg tcgctgctca cgtccggctt cc#agctggat   4080gaccccaccg gctacgccga gcgcatcaac cgcatgatca agctcggcct gt#cgctcgac   4140gaggaggagg aggaggtcgc cgaggcgccg ccggccgagg cagcccccgc gg#aggtcacc   4200 gccggcacct ccagcatgga gcaggtggac taa       #                   #       4233 <210> SEQ ID NO 100 <211> LENGTH: 4917<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: DNA sequence encoding fus #ion (poly-protein)      constructs comprising multiple Leishman #ia antigens<400> SEQUENCE: 100ccagtgtggt ggatgtcctg cggtaacgcc aagatcaact ctcccgcgcc gt#ccttcgag     60gaggtggcgc tcatgcccaa cggcagcttc aagaagatca gcctctcctc ct#acaagggc    120aagtgggtcg tgctcttctt ctacccgctc gacttcacct tcgtgtgccc ga#cagaggtc    180atcgcgttct ccgacagcgt gagtcgcttc aacgagctca actgcgaggt cc#tcgcgtgc    240tcgatagaca gcgagtacgc gcacctgcag tggacgctgc aggaccgcaa ga#agggcggc    300ctcgggacca tggcgatccc aatgctagcc gacaagacca agagcatcgc tc#gttcctac    360ggcgtgctgg aggagagcca gggcgtggcc taccgcggtc tcttcatcat cg#acccccat    420ggcatgctgc gtcagatcac cgtcaatgac atgccggtgg gccgcagcgt gg#aggaggtt    480ctacgcctgc tggaggcttt tcagttcgtg gagaagcacg gcgaggtgtg cc#ccgcgaac    540tggaagaagg gcgcccccac gatgaagccg gaaccgaatg cgtctgtcga gg#gatacttc    600agcaagcagg gatccatgga cgcaactgag ctgaagaaca aggggaacga ag#agttctcc    660gccggccgct atgtggaggc ggtgaactac ttctcaaagg cgatccagtt gg#atgagcag    720aacagtgtcc tctacagcaa ccgctccgcc tgttttgcag ccatgcagaa at#acaaggac    780gcgctggacg acgccgacaa gtgcatctcg atcaagccga attgggccaa gg#gctacgtg    840cgccgaggag cagctctcca tggcatgcgc cgctacgacg atgccattgc cg#cgtatgaa    900aaggggctca aggtggaccc ttccaacagc ggctgcgcgc agggcgtgaa gg#acgtgcag    960gtagccaagg cccgcgaagc acgtgacccc atcgctcgcg tcttcacccc gg#aggcgttc   1020cgcaagatcc aagagaatcc caagctgtct ctacttatgc tgcagccgga ct#acgtgaag   1080atggtagaca ccgtcatccg cgacccttcg cagggccggc tgtacatgga ag#accagcgc   1140tttgccctga cgctcatgta cctgagcgga atgaagattc ccaacgatgg tg#atggcgag   1200gaggaggaac gtccgtctgc gaaggcggca gagacagcga agccaaaaga gg#agaagcct   1260ctcaccgaca acgagaagga ggccctggcg ctcaaggagg agggcaacaa gc#tgtacctc   1320tcgaagaagt ttgaggaggc gctgaccaag taccaagagg cgcaggtgaa ag#accccaac   1380aacactttat acattctgaa cgtgtcggcc gtgtacttcg agcagggtga ct#acgacaag   1440tgcatcgccg agtgcgagca cggtatcgag cacggtcgcg agaaccactg cg#actacaca   1500atcattgcga agctcatgac ccggaacgcc ttgtgcctcc agaggcagag ga#agtacgag   1560gctgctatcg acctttacaa gcgcgccctt gtcgagtggc gtaaccctga ca#ccctcaag   1620aagctgacgg agtgcgagaa ggagcaccaa aaggcggtgg aggaagccta ca#tcgatcct   1680gagatcgcga agcagaagaa agacgaaggt aaccagtact tcaaggagga ta#agttcccc   1740gaggccgtgg cagcgtacac ggaggccatc aagcgcaacc ctgccgagca ca#cctcctac   1800agcaatcgcg cggccgcgta catcaagctt ggagccttca acgacgccct ca#aggacgcg   1860gagaagtgca ttgagctgaa gcccgacttt gttaagggct acgcgcgcaa gg#gtcatgct   1920tacttttgga ccaagcagta caaccgcgcg ctgcaggcgt acaatgaggg cc#tcaaggtg   1980gacccgagca atgcggactg caaggatggg cggtatcgca caatcatgaa ga#ttcaggag   2040atggcatctg gccaatccgc ggatggcgac gaggcggcgc gccgggccat gg#acgatcct   2100gaaatcgcgg caatcatgca agatagctac atgcaactag tgttgaagga ga#tgcagaac   2160gatcccacgc gcattcagga gtacatgaag gactccggga tctcatcgaa ga#tcaacaag   2220ctgatttcag ctggcatcat tcgttttggt caggaattca gcctgacgga cc#cggcggtg   2280ctgggcgagg agactcacct gcgcgtccgc gtggtgccgg acaaggcgaa ca#agacgctg   2340acggtggagg ataacggcat cggcatgacc aaggcggacc tcgtgaacaa tc#tgggcacg   2400atcgcgcgct ccggcacgaa ggcgttcatg gaggcactgg aggccggcgg cg#acatgagc   2460atgatcggcc agttcggtgt cggcttctac tccgcgtacc ttgtggcgga cc#gcgtgacg   2520gtggtgtcga agaacaactc ggacgaggcg tacgtatggg agtcgtccgc gg#gcggcacg   2580ttcaccatca cgagcgtgcc ggagtcggac atgaagcgcg gcacgcgcat ca#cgctgcac   2640ctaaaggagg accagcagga gtacctggag gagcgccggg tgaaggagct ga#tcaagaag   2700cactccgagt tcatcggcta cgacatcgag ctgatggtgg agaagacggc gg#agaaggag   2760gtgacggacg aggacgagga ggaggacgag tcgaagaaga agtcctgcgg gg#acgagggc   2820gagccgaagg tggaggaggt gacggagggc ggcgaggaca agaagaagaa ga#cgaagaag   2880gtgaaggagg tgacgaagac gtacgaggtc cagaacaagc acaagccgct ct#ggacgcgc   2940gacccgaagg acgtgacgaa ggaggagtac gcggccttct acaaggccat ct#ccaacgac   3000tgggaggacc cggcggcgac gaagcacttc tcggtggagg gccagctgga gt#tccgcgcg   3060atcgcgttcg tgccgaagcg cgcgccgttc gacatgttcg agccgaacaa ga#agcgcaac   3120aacatcaagc tgtacgtgcg ccgcgtgttc atcatggaca actgcgagga cc#tgtgcccg   3180gactggctcg gcttcgtgaa gggcgtcgtg gacagcgagg acctgccgct ga#acatctcg   3240cgcgagaacc tgcagcagaa caagatcctg aaggtgatcc gcaagaacat cg#tgaagaag   3300tgcctggagc tgttcgaaga gatagcggag aacaaggagg actacaagca gt#tctacgag   3360cagttcggca agaacatcaa gctgggcatc cacgaggaca cggcgaaccg ca#agaagctg   3420atggagttgc tgcgcttcta cagcaccgag tcgggggagg agatgacgac ac#tgaaggac   3480tacgtgacgc gcatgaagcc ggagcagaag tcgatctact acatcactgg cg#acagcaag   3540aagaagctgg agtcgtcgcc gttcatcgag aaggcgagac gctgcgggct cg#aggtgctg   3600ttcatgacgg agccgatcga cgagtacgtg atgcagcagg tgaaggactt cg#aggacaag   3660aagttcgcgt gcctgacgaa ggaaggcgtg cacttcgagg agtccgagga gg#agaagaag   3720cagcgcgagg agaagaaggc ggcgtgcgag aagctgtgca agacgatgaa gg#aggtgctg   3780ggcgacaagg tggagaaggt gaccgtgtcg gagcgcctgt cgacgtcgcc gt#gcatcctg   3840gtgacgtcgg agtttgggtg gtcggcgcac atggaacaga tcatgcgcaa cc#aggcgctg   3900cgcgactcca gcatggcgca gtacatggtg tccaagaaga cgatggaggt ga#accccgac   3960caccccatca tcaaggagct gcgccgccgc gtggaggcgg acgagaacga ca#aggccgtg   4020aaggacctcg tcttcctgct cttcgacacg tcgctgctca cgtccggctt cc#agctggat   4080gaccccaccg gctacgccga gcgcatcaac cgcatgatca agctcggcct gt#cgctcgac   4140gaggaggagg aggaggtcgc cgaggcgccg ccggccgagg cagcccccgc gg#aggtcacc   4200gccggcacct ccagcatgga gcaggtggac gatatcatgg cgcagaatga ta#agatcgcc   4260ccccaggacc aggactcctt cctcgatgac cagcccggcg ttcgcccgat cc#cgtccttc   4320gacgacatgc cgctgcacca gaacctgctg cgtggcatct actcgtacgg gt#tcgagaag   4380ccgtccagca tccagcagcg cgcgatagcc cccttcacgc gcggcggcga ca#tcatcgcg   4440caggcccagt ccggtaccgg caagacgggt gccttctcca tcggtctgct gc#agcgcctg   4500gacttccgcc acaacctgat ccagggcctc gtgctctccc ccactcgcga gc#tggccctg   4560cagacggcgg aggtgatcag ccgcatcggt gagttcctgt cgaacagctc ca#agttctgc   4620gagacctttg tcggcggcac gcgcgtgcag gatgacctgc gcaagctgca gg#ccggcgtc   4680atcgttgccg tgggcacgcc gggccgcgtg tccgacgtga tcaagcgtgg cg#cgctgcgc   4740acagagtcgc tgcgcgtgct ggtgctcgac gaggctgatg agatgctgtc tc#agggcttc   4800gcggaccaga tttacgagat cttccgcttc ctgccgaagg acatccaggt cg#cgctcttc   4860tccgccacga tgccggagga ggtactggag ctgacgaaga agttcatgcg cg#actaa      4917 <210> SEQ ID NO 101 <211> LENGTH: 2735 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: DNA sequence encoding fus #ion (poly-protein)      constructs comprising multiple Leishman #ia antigens<400> SEQUENCE: 101catatgcacc accaccacca ccacatgtcc tgcggtaacg ccaagatcaa ct#ctcccgcg     60ccgtccttcg aggaggtggc gctcatgccc aacggcagct tcaagaagat ca#gcctctcc    120tcctacaagg gcaagtgggt cgtgctcttc ttctacccgc tcgacttcac ct#tcgtgtgc    180ccgacagagg tcatcgcgtt ctccgacagc gtgagtcgct tcaacgagct ca#actgcgag    240gtcctcgcgt gctcgataga cagcgagtac gcgcacctgc agtggacgct gc#aggaccgc    300aagaagggcg gcctcgggac catggcgatc ccaatgctag ccgacaagac ca#agagcatc    360gctcgttcct acggcgtgct ggaggagagc cagggcgtgg cctaccgcgg tc#tcttcatc    420atcgaccccc atggcatgct gcgtcagatc accgtcaatg acatgccggt gg#gccgcagc    480gtggaggagg ttctacgcct gctggaggct tttcagttcg tggagaagca cg#gcgaggtg    540tgccccgcga actggaagaa gggcgccccc acgatgaagc cggaaccgaa tg#cgtctgtc    600gagggatact tcagcaagca gggatccatg gacgcaactg agctgaagaa ca#aggggaac    660gaagagttct ccgccggccg ctatgtggag gcggtgaact acttctcaaa gg#cgatccag    720ttggatgagc agaacagtgt cctctacagc aaccgctccg cctgttttgc ag#ccatgcag    780aaatacaagg acgcgctgga cgacgccgac aagtgcatct cgatcaagcc ga#attgggcc    840aagggctacg tgcgccgagg agcagctctc catggcatgc gccgctacga cg#atgccatt    900gccgcgtatg aaaaggggct caaggtggac ccttccaaca gcggctgcgc gc#agggcgtg    960aaggacgtgc aggtagccaa ggcccgcgaa gcacgtgacc ccatcgctcg cg#tcttcacc   1020ccggaggcgt tccgcaagat ccaagagaat cccaagctgt ctctacttat gc#tgcagccg   1080gactacgtga agatggtaga caccgtcatc cgcgaccctt cgcagggccg gc#tgtacatg   1140gaagaccagc gctttgccct gacgctcatg tacctgagcg gaatgaagat tc#ccaacgat   1200ggtgatggcg aggaggagga acgtccgtct gcgaaggcgg cagagacagc ga#agccaaaa   1260gaggagaagc ctctcaccga caacgagaag gaggccctgg cgctcaagga gg#agggcaac   1320aagctgtacc tctcgaagaa gtttgaggag gcgctgacca agtaccaaga gg#cgcaggtg   1380aaagacccca acaacacttt atacattctg aacgtgtcgg ccgtgtactt cg#agcagggt   1440gactacgaca agtgcatcgc cgagtgcgag cacggtatcg agcacggtcg cg#agaaccac   1500tgcgactaca caatcattgc gaagctcatg acccggaacg ccttgtgcct cc#agaggcag   1560aggaagtacg aggctgctat cgacctttac aagcgcgccc ttgtcgagtg gc#gtaaccct   1620gacaccctca agaagctgac ggagtgcgag aaggagcacc aaaaggcggt gg#aggaagcc   1680tacatcgatc ctgagatcgc gaagcagaag aaagacgaag gtaaccagta ct#tcaaggag   1740gataagttcc ccgaggccgt ggcagcgtac acggaggcca tcaagcgcaa cc#ctgccgag   1800cacacctcct acagcaatcg cgcggccgcg tacatcaagc ttggagcctt ca#acgacgcc   1860ctcaaggacg cggagaagtg cattgagctg aagcccgact ttgttaaggg ct#acgcgcgc   1920aagggtcatg cttacttttg gaccaagcag tacaaccgcg cgctgcaggc gt#acaatgag   1980ggcctcaagg tggacccgag caatgcggac tgcaaggatg ggcggtatcg ca#caatcatg   2040aagattcagg agatggcatc tggccaatcc gcggatggcg acgaggcggc gc#gccgggcc   2100atggacgatc ctgaaatcgc ggcaatcatg caagatagct acatgcaact ag#tgttgaag   2160gagatgcaga acgatcccac gcgcattcag gagtacatga aggactccgg ga#tctcatcg   2220aagatcaaca agctgatttc agctggcatc attcgttttg gtcaggaatt ct#gcagatat   2280ccatcacact ggcggccgct cgagcagatc cggctgctaa caaagcccga aa#ggaagctg   2340agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tc#taaacggg   2400tcttgagggg ttttttgctg aaaggaggaa ctatatccgg ataattcttg aa#gacgaaag   2460ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt tt#cttagacg   2520tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttggttatt tt#tctaaata   2580cattcaaata tgtatccgct catgagacaa taacccytga taaatgcttc aa#taatattg   2640aaaaaaggaa gaatatgaag tatttcaaca tttcccgggt cccccttatt cc#cttttttt   2700 gccgccattt tgcctttctg tttttggttc accca       #                   #     2735 <210> SEQ ID NO 102 <211> LENGTH: 1713<212> TYPE: DNA <213> ORGANISM: Leishmania major <400> SEQUENCE: 102atggcgcagt gcgtgcgtcg gctggtgctg gcggcgacgc tcgccgctgc gg#tggcgctg     60ctgctgtgca cgagcagtgc gccggtggcg cgtgctgctg ggacgaacga ct#tcactgcg    120gcgcagcgga cgaacacgct ggcggtgctg caggcgtttg ggcgtgcgat cc#ctgagctt    180ggggagaagt gggcgggcaa cgacttctgc tcatgggagt ttatcgtgtg ta#atgttata    240ggtgtgaacg tacggggaat cagtccgacg tatgccggca cgctgccgga ga#tacctgtg    300aacgtcgact acaggcacgt cgtgatcaag cagctcgact tttccgaaat gg#ggccgggg    360ctgagcggga cgctgccgga cagctggagc aagctggaag gactgacttc cc#ttacgttg    420tcgggcaaca aagtgagcgg tacgctgccc gcctcatggc acttgatgaa gc#ggttgaca    480tctttggtaa ttgcagactt tgacagtatc accggcagcc tgccgcctga gt#ggagctcg    540atgcctaatt taaacgctgt ggagctgaag cgactaaaac tgagcggtac gt#tgcctgcg    600gactggagct ctttgaaatc actgtcgaac gtcgttcttg aggacacgcc ga#tcacaggc    660ttgttgcccc cggagtgggc ctcgctggag agaatacagc agctggttct ac#ggaaattg    720aagctgaccg gccctctccc tcctcagtgg agctcaatga agatattgca gt#atcttact    780ctggatggca ctcaggtctc cggcacgctg ccgccccagt ggagcgcgat gg#catcggtg    840cgaattctta acctggaggg tactgaggtc tctggtacgc tgccgcctga gt#ggatatcg    900atgagcaggc tgcaaactct gaatctgcgg cgcacgaaag tatccggcac tc#tgccgccc    960gaatggagtt ctatgagcag cctggagtac tttcaccttt atcttactca gg#tctccggc   1020acgctgccgc ccgagtggag tgggatgtcg aaggccgcat acttctggct gg#aatactgc   1080gacctgtccg gcagtctgcc gcccgagtgg tcgtcgatgc caaagctgcg cg#gtatctca   1140ctgagcggca acaagttctg cgggtgtgtg ccggactcgt gggatcagaa gg#ctggtctt   1200gttgtgggca tcgaggacaa gcacaagggc agcgactgct tggctgctaa gg#actgcaca   1260acgaccacca caaaaccccc caccacgaca acgaccccca ctaagccgcc tg#ccacaacc   1320accactgagg caccggctga acccacgacc accactgagg caccggctga ac#ccacgacc   1380accactgagg caccggctga acccacgacc accactgagg caccggctga ac#ccacaacc   1440accactgagg caccggctga acccacgacc actgctaccc caacaaacac gc#cgactcct   1500gcaccagaga cggagtgcga ggtggatggg tgtgaggtgt gcgaggggga ct#ccgctgcg   1560aggtgcgcga ggtgccgtga ggactacttc ctgacggacg agaggacgtg cc#tggtgtac   1620tgcgatggcg gtgttgctgc tgtgtcgagc ggagtggcag cagcagctgt tg#tgtgcgtg   1680 gctgtgctgt tcagcgtggg gctggcggcg tga       #                   #       1713 <210> SEQ ID NO 103 <211> LENGTH: 2421<212> TYPE: DNA <213> ORGANISM: Leishmania major <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(2421)<223> OTHER INFORMATION: n=A,T,C or G <400> SEQUENCE: 103tcgaattcgg cacgagggca cgcacaagac gaagagttcc aaacagcaac ga#gtatacgc     60cactgtcgaa aaacagacac gcagtagaga gaaggaggag gaggaggagg ag#gggggaga    120gcaagaggcg ggtgggggtt ggagggacag cgctgcgtgc cgccgtctga ca#tgtccgtt    180ttgatgcgtc ttcgcagagt ggagggagga caccactggc gctgttggcg tg#tangcaga    240gcatcgctcg gctcgtgccg aattcggcac gagcggcacg agccctcgct ct#gcctggta    300agctcagcag acaccgacgc ccgagcaatc ccgcccacgg acctgctgcc gc#cccgctct    360gctcgtgacc ctggctgcga atggcgcagt gcgtgcgtcg gctggtgctg gc#ggcgacgc    420tcgccgctgc ggtggcgctg ctgctgtgca cgagcagtgc gccggtggcg cg#tgctgctg    480ggacgaacga cttcactgcg gcgcagcgga cgaacacgct ggcggtgctg ca#ggcgtttg    540ggcgtgcgat ccctgagctt ggggagaagt gggcgggcaa cgacttctgc tc#atgggagt    600ttatcgtgtg taatgttata ggtgtgaacg tacggggaat cagtccgacg ta#tgccggca    660cgctgccgga gatacctgtg aacgtcgact acaggcacgt cgtgatcaag ca#gctcgact    720tttccgaaat ggggccgggg ctgagcggga cgctgccgga cagctggagc aa#gctggaag    780gactgacttc ccttacgttg tcgggcaaca aagtgagcgg tacgctgccc gc#ctcatggc    840acttgatgaa gcggttgaca tctttggtaa ttgcagactt tgacagtatc ac#cggcagcc    900tgccgcctga gtggagctcg atgcctaatt taaacgctgt ggagctgaag cg#actaaaac    960tgagcggtac gttgcctgcg gactggagct ctttgaaatc actgtcgaac gt#cgttcttg   1020aggacacgcc gatcacaggc ttgttgcccc cggagtgggc ctcgctggag ag#aatacagc   1080agctggttct acggaaattg aagctgaccg gccctctccc tcctcagtgg ag#ctcaatga   1140agatattgca gtatcttact ctggatggca ctcaggtctc cggcacgctg cc#gccccagt   1200ggagcgcgat ggcatcggtg cgaattctta acctggaggg tactgaggtc tc#tggtacgc   1260tgccgcctga gtggatatcg atgagcaggc tgcaaactct gaatctgcgg cg#cacgaaag   1320tatccggcac tctgccgccc gaatggagtt ctatgagcag cctggagtac tt#tcaccttt   1380atcttactca ggtctccggc acgctgccgc ccgagtggag tgggatgtcg aa#ggccgcat   1440acttctggct ggaatactgc gacctgtccg gcagtctgcc gcccgagtgg tc#gtcgatgc   1500caaagctgcg cggtatctca ctgagcggca acaagttctg cgggtgtgtg cc#ggactcgt   1560gggatcagaa ggctggtctt gttgtgggca tcgaggacaa gcacaagggc ag#cgactgct   1620tggctgctaa ggactgcaca acgaccacca caaaaccccc caccacgaca ac#gaccccca   1680ctaagccgcc tgccacaacc accactgagg caccggctga acccacgacc ac#cactgagg   1740caccggctga acccacgacc accactgagg caccggctga acccacgacc ac#cactgagg   1800caccggctga acccacaacc accactgagg caccggctga acccacgacc ac#tgctaccc   1860caacaaacac gccgactcct gcaccagaga cggagtgcga ggtggatggg tg#tgaggtgt   1920gcgaggggga ctccgctgcg aggtgcgcga ggtgccgtga ggactacttc ct#gacggacg   1980agaggacgtg cctggtgtac tgcgatggcg gtgttgctgc tgtgtcgagc gg#agtggcag   2040cagcagctgt tgtgtgcgtg gctgtgctgt tcagcgtggg gctggcggcg tg#aggacgct   2100gctgctgttg cgcgcaggca gcggcccccg ctgcgtggca cacgactgtc tg#cgtgcttg   2160cgtgcagcgc cgccccctgc gttggcgtgc gcgtgcgtgt ctctgtgagc at#ggctgcca   2220gtggtgccct cgctcctgcc tctcggtgcc tctgcctctc tcggcgtgtt ga#tgctgtgg   2280gctgtgtgtg gggctctcat gcggcgctgc tgctcccgcg gtgtcgctcn tc#tgccccga   2340ctctctctgc tgccctcctc tctcgcatgc gggagaggga ggggtggcac gt#gcgcgcgc   2400 gcmgttgcgc ttgcgattgt g            #                  #                2421 <210> SEQ ID NO 104 <211> LENGTH: 570<212> TYPE: PRT <213> ORGANISM: Leishmania major <400> SEQUENCE: 104Met Ala Gln Cys Val Arg Arg Leu Val Leu Al #a Ala Thr Leu Ala Ala                   #5                   #10                   #15Ala Val Ala Leu Leu Leu Cys Thr Ser Ser Al #a Pro Val Ala Arg Ala             20      #             25      #             30Ala Gly Thr Asn Asp Phe Thr Ala Ala Gln Ar #g Thr Asn Thr Leu Ala         35          #         40          #         45Val Leu Gln Ala Phe Gly Arg Ala Ile Pro Gl #u Leu Gly Glu Lys Trp     50              #     55              #     60Ala Gly Asn Asp Phe Cys Ser Trp Glu Phe Il #e Val Cys Asn Val Ile 65                  # 70                  # 75                  # 80Gly Val Asn Val Arg Gly Ile Ser Pro Thr Ty #r Ala Gly Thr Leu Pro                 85  #                 90  #                 95Glu Ile Pro Val Asn Val Asp Tyr Arg His Va #l Val Ile Lys Gln Leu            100       #           105       #           110Asp Phe Ser Glu Met Gly Pro Gly Leu Ser Gl #y Thr Leu Pro Asp Ser        115           #       120           #       125Trp Ser Lys Leu Glu Gly Leu Thr Ser Leu Th #r Leu Ser Gly Asn Lys    130               #   135               #   140Val Ser Gly Thr Leu Pro Ala Ser Trp His Le #u Met Lys Arg Leu Thr145                 1 #50                 1 #55                 1 #60Ser Leu Val Ile Ala Asp Phe Asp Ser Ile Th #r Gly Ser Leu Pro Pro                165   #               170   #               175Glu Trp Ser Ser Met Pro Asn Leu Asn Ala Va #l Glu Leu Lys Arg Leu            180       #           185       #           190Lys Leu Ser Gly Thr Leu Pro Ala Asp Trp Se #r Ser Leu Lys Ser Leu        195           #       200           #       205Ser Asn Val Val Leu Glu Asp Thr Pro Ile Th #r Gly Leu Leu Pro Pro    210               #   215               #   220Glu Trp Ala Ser Leu Glu Arg Ile Gln Gln Le #u Val Leu Arg Lys Leu225                 2 #30                 2 #35                 2 #40Lys Leu Thr Gly Pro Leu Pro Pro Gln Trp Se #r Ser Met Lys Ile Leu                245   #               250   #               255Gln Tyr Leu Thr Leu Asp Gly Thr Gln Val Se #r Gly Thr Leu Pro Pro            260       #           265       #           270Gln Trp Ser Ala Met Ala Ser Val Arg Ile Le #u Asn Leu Glu Gly Thr        275           #       280           #       285Glu Val Ser Gly Thr Leu Pro Pro Glu Trp Il #e Ser Met Ser Arg Leu    290               #   295               #   300Gln Thr Leu Asn Leu Arg Arg Thr Lys Val Se #r Gly Thr Leu Pro Pro305                 3 #10                 3 #15                 3 #20Glu Trp Ser Ser Met Ser Ser Leu Glu Tyr Ph #e His Leu Tyr Leu Thr                325   #               330   #               335Gln Val Ser Gly Thr Leu Pro Pro Glu Trp Se #r Gly Met Ser Lys Ala            340       #           345       #           350Ala Tyr Phe Trp Leu Glu Tyr Cys Asp Leu Se #r Gly Ser Leu Pro Pro        355           #       360           #       365Glu Trp Ser Ser Met Pro Lys Leu Arg Gly Il #e Ser Leu Ser Gly Asn    370               #   375               #   380Lys Phe Cys Gly Cys Val Pro Asp Ser Trp As #p Gln Lys Ala Gly Leu385                 3 #90                 3 #95                 4 #00Val Val Gly Ile Glu Asp Lys His Lys Gly Se #r Asp Cys Leu Ala Ala                405   #               410   #               415Lys Asp Cys Thr Thr Thr Thr Thr Lys Pro Pr #o Thr Thr Thr Thr Thr            420       #           425       #           430Pro Thr Lys Pro Pro Ala Thr Thr Thr Thr Gl #u Ala Pro Ala Glu Pro        435           #       440           #       445Thr Thr Thr Thr Glu Ala Pro Ala Glu Pro Th #r Thr Thr Thr Glu Ala    450               #   455               #   460Pro Ala Glu Pro Thr Thr Thr Thr Glu Ala Pr #o Ala Glu Pro Thr Thr465                 4 #70                 4 #75                 4 #80Thr Thr Glu Ala Pro Ala Glu Pro Thr Thr Th #r Ala Thr Pro Thr Asn                485   #               490   #               495Thr Pro Thr Pro Ala Pro Glu Thr Glu Cys Gl #u Val Asp Gly Cys Glu            500       #           505       #           510Val Cys Glu Gly Asp Ser Ala Ala Arg Cys Al #a Arg Cys Arg Glu Asp        515           #       520           #       525Tyr Phe Leu Thr Asp Glu Arg Thr Cys Leu Va #l Tyr Cys Asp Gly Gly    530               #   535               #   540Val Ala Ala Val Ser Ser Gly Val Ala Ala Al #a Ala Val Val Cys Val545                 5 #50                 5 #55                 5 #60Ala Val Leu Phe Ser Val Gly Leu Ala Ala                 565  #               570 <210> SEQ ID NO 105 <211> LENGTH: 1688<212> TYPE: DNA <213> ORGANISM: Leishmania major <400> SEQUENCE: 105taacgctata taagtatcag tttctgtact ttattgctca tcactgccgt tt#gactgccg     60cgggcattgg cgctaccact ttcctcactc tttatccctt cagcattgtt tc#gtacacac    120gcacgcgcac gtgaaagagc cgcacgccga cagagcagcc gttccggact ct#ccgataac    180tgaacgccac ccacccaaaa aaaatgtcaa agaacgctga ccaggaggag tg#ggaggatt    240acggcgacga ggaggtgcag gatgaagaag aggaggacac caccatcaac aa#ctccgacg    300tggtggtgcg ctacaagaag gccgcaacgt ggtgcaatga aacgttgcgc gt#gcttatcg    360atgccacaaa acctggcgcc aaggtgtgcg acctgtgccg cctcggtgat ga#caccatca    420ccgccaaggt caagacaatg ttcaaaggca cggaaaaagg catcgctttc cc#gacctgca    480tctcggtcaa caactgcgta tgccacaaca gccctggcgt gtcggacgag ac#gacgcagc    540aagagatcgc gatgggtgac gtcgtgcact acgacctggg catccacgtg ga#cggctact    600gcgccgtcgt cgcgcacacc attcaggtga cagaggacaa tgagcttggc aa#ggacgaga    660aggcggcgcg cgtcattaca gcggcgtaca acatcctgaa cacggcgctg cg#ccagatgc    720gtcccggtac gaccatctac caggtgacag acgtagttga gaaggctgcg ga#gcactaca    780aggtgactcc ggtagacggc gtcctctcgc atatgatgaa gcgctacatc at#agacggat    840accgctgtat cccgcagcgc agggtcgcgg agcacatggt gcacgactac ga#tctcgaga    900aagcgcaggt gtggacgcta gacattgtca tgacctccgg caagggcaag ct#gaaggagc    960gcgatgcgcg gccgtgcgtg ttcaaggtgg ctctggactc caactactct gt#gaaaatgg   1020aaagcgcgaa ggaggttcag aaggaaatcg actccaagta tgccaccttc cc#ctttgcca   1080tccgcaacct ggaggccaag aaggcccgcc tcggtctcaa cgagatggcg aa#gcacggtg   1140ctgtcatccc gtaccctatt ctcttcgaaa aggaaggcga ggtcgtcgcc ca#tttcaaga   1200ttacggtgct catcagcaac aagaagattg agccgattac cggcctgaag cc#gcagaagg   1260ccccggcgct cgagccatac acggacgaga tgctgcttgc gacgaacaag ct#ctcgctgt   1320cgctagagaa gaaggcggcg aagtagacgg ccgtggcatc cgtgacgctg ta#ctgcgagc   1380tttcgtaggc gtacgcctct tgtgaggcgt acacgtgtgc tgtttgcgga cg#aggaggca   1440cccattctgt tccccttctt cgctaatctc cgcgtttcct ctgacgctgg ct#tctctgcc   1500ggagtgtggt gaggcgcgtg ggggagaaac ggcccactcg catgcctgtg ca#tacgcgag   1560cacggtaggg agcgcggtgt gtgtgtgtgt gggggggcgt gttacgagta ca#aaagaggc   1620tcgatctctg cgactctttt ctttctgtaa acagggaaca taagtaacca aa#aaaaaaaa   1680 aaaaaaaa                 #                  #                   #        1688 210> SEQ ID NO 106 <211> LENGTH: 380<212> TYPE: PRT <213> ORGANISM: Leishmania major <400> SEQUENCE: 106Met Ser Lys Asn Ala Asp Gln Glu Glu Trp Gl #u Asp Tyr Gly Asp Glu 1               5   #                10   #                15Glu Val Gln Asp Glu Glu Glu Glu Asp Thr Th #r Ile Asn Asn Ser Asp            20       #            25       #            30Val Val Val Arg Tyr Lys Lys Ala Ala Thr Tr #p Cys Asn Glu Thr Leu        35           #        40           #        45Arg Val Leu Ile Asp Ala Thr Lys Pro Gly Al #a Lys Val Cys Asp Leu    50               #    55               #    60Cys Arg Leu Gly Asp Asp Thr Ile Thr Ala Ly #s Val Lys Thr Met Phe65                   #70                   #75                   #80Lys Gly Thr Glu Lys Gly Ile Ala Phe Pro Th #r Cys Ile Ser Val Asn                85   #                90   #                95Asn Cys Val Cys His Asn Ser Pro Gly Val Se #r Asp Glu Thr Thr Gln            100       #           105       #           110Gln Glu Ile Ala Met Gly Asp Val Val His Ty #r Asp Leu Gly Ile His        115           #       120           #       125Val Asp Gly Tyr Cys Ala Val Val Ala His Th #r Ile Gln Val Thr Glu    130               #   135               #   140Asp Asn Glu Leu Gly Lys Asp Glu Lys Ala Al #a Arg Val Ile Thr Ala145                 1 #50                 1 #55                 1 #60Ala Tyr Asn Ile Leu Asn Thr Ala Leu Arg Gl #n Met Arg Pro Gly Thr                165   #               170   #               175Thr Ile Tyr Gln Val Thr Asp Val Val Glu Ly #s Ala Ala Glu His Tyr            180       #           185       #           190Lys Val Thr Pro Val Asp Gly Val Leu Ser Hi #s Met Met Lys Arg Tyr        195           #       200           #       205Ile Ile Asp Gly Tyr Arg Cys Ile Pro Gln Ar #g Arg Val Ala Glu His    210               #   215               #   220Met Val His Asp Tyr Asp Leu Glu Lys Ala Gl #n Val Trp Thr Leu Asp225                 2 #30                 2 #35                 2 #40Ile Val Met Thr Ser Gly Lys Gly Lys Leu Ly #s Glu Arg Asp Ala Arg                245   #               250   #               255Pro Cys Val Phe Lys Val Ala Leu Asp Ser As #n Tyr Ser Val Lys Met            260       #           265       #           270Glu Ser Ala Lys Glu Val Gln Lys Glu Ile As #p Ser Lys Tyr Ala Thr        275           #       280           #       285Phe Pro Phe Ala Ile Arg Asn Leu Glu Ala Ly #s Lys Ala Arg Leu Gly    290               #   295               #   300Leu Asn Glu Met Ala Lys His Gly Ala Val Il #e Pro Tyr Pro Ile Leu305                 3 #10                 3 #15                 3 #20Phe Glu Lys Glu Gly Glu Val Val Ala His Ph #e Lys Ile Thr Val Leu                325   #               330   #               335Ile Ser Asn Lys Lys Ile Glu Pro Ile Thr Gl #y Leu Lys Pro Gln Lys            340       #           345       #           350Ala Pro Ala Leu Glu Pro Tyr Thr Asp Glu Me #t Leu Leu Ala Thr Asn        355           #       360           #       365Lys Leu Ser Leu Ser Leu Glu Lys Lys Ala Al #a Lys     370              #   375               #   380 <210> SEQ ID NO 107 <211> LENGTH: 1565<212> TYPE: DNA <213> ORGANISM: Leishmania major <400> SEQUENCE: 107taacgctata taagtatcag tttctgtact ttattgctct tcgctctcgt tc#ttcgaaca     60aacaccttta aaccgccttc caacccctct ttcttctttt tcagccatgc gt#gaggctat    120ctgcatccac atcggccagg ccggctgcca ggtcggtaac gcgtgctggg ag#ctgttctg    180ccttgagcac ggcatccagc ctgatggctc catgccctct gacaagtgca tc#ggtgttga    240ggatgacgcg ttcaacacgt tcttctcgga gaccggtgct ggcaagcacg tt#ccgcgctg    300catcttcctg gacctcgagc ctacggtcgt ggatgaggtg cgcaccggca cg#taccgcca    360gctgttcaac cccgagcagc tggtgtctgg caaggaggat gcggcgaaca ac#tacgctcg    420tggccactac acgatcggca aggagatcgt cgaccttgcg ctggaccgca tt#cgcaagct    480ggcggacaac tgcactggtc tccagggctt tatggtgttc cacgctgtgg gt#ggcggcac    540cggctctggc ctcggtgcgc tgctgctgga gcgcctgtct gtggactacg gc#aagaagtc    600caagctcggc tacaccgtgt acccgagccc gcaggtgtcg actgccgtcg tg#gagccgta    660caactgcgtg ctgtcgacgc actcgctgct cgagcacacc gatgttgcga cg#atgctcga    720caatgaggcc atctacgacc tcactcgtcg ttctctcgac attgagcgcc cg#tcgtacac    780gaacgtgaac cgcctgatcg gccaggtggt gtcgtctctg acggcgtcgc tg#cgcttcga    840tggtgcgctg aacgtggacc tgacggagtt ccagacgaac cttgtgccgt ac#ccgcgcat    900ccacttcgtg ctgacgagct acgctccggt ggtgtctgcc gagaaggcgt ac#cacgagca    960gctgtccgtc gcggacatca cgaactcggt gtttgagcct gctggcatgc tg#acgaagtg   1020cgatcctcgc cacggcaagt acatgtcgtg ctgcctcatg taccgcggtg at#gtcgtgcc   1080gaaggatgtc aacgccgcga ttgcgacgat caagacgaag cgcacaattc ag#ttcgtgga   1140ctggtgcccg accggcttca agtgcggcat caactaccag ccgccgaccg tt#gtgcccgg   1200cggtgacctc gcgaaggtgc agcgcgccgt gtgcatgatt gccaactcga cc#gcgatcgc   1260tgaggtgttt gcccgcatcg accacaagtt cgacctgatg tacagcaagc gc#gcgtttgt   1320gcactggtac gtgggtgagg gcatggagga gggcgagttc tccgaggcgc gc#gaggatct   1380cgctgcgctg gagaaggact acgaggaggt tggcgccgag tccgccgacg ac#atgggcga   1440ggaggacgtc gaggagtact aaggtagact cgtgccgcgc gctgatgatg ta#ggtgcacg   1500cgtgcgtgtg ctgcagcgga gccgccgcca ccgcgactgt gtgtgtgtgc gc#gcgtgacg   1560 accgg                  #                  #                   #          1565 <210> SEQ ID NO 108<211> LENGTH: 451 <212> TYPE: PRT <213> ORGANISM: Leishmania major<400> SEQUENCE: 108 Met Arg Glu Ala Ile Cys Ile His Ile Gly Gl#n Ala Gly Cys Gln Val  1               5   #                10  #                15 Gly Asn Ala Cys Trp Glu Leu Phe Cys Leu Gl#u His Gly Ile Gln Pro             20       #            25      #            30 Asp Gly Ser Met Pro Ser Asp Lys Cys Ile Gl#y Val Glu Asp Asp Ala         35           #        40          #        45 Phe Asn Thr Phe Phe Ser Glu Thr Gly Ala Gl#y Lys His Val Pro Arg     50               #    55              #    60 Cys Ile Phe Leu Asp Leu Glu Pro Thr Val Va#l Asp Glu Val Arg Thr 65                   #70                  #75                   #80 Gly Thr Tyr Arg Gln Leu Phe Asn Pro Glu Gl#n Leu Val Ser Gly Lys                 85   #                90  #                95 Glu Asp Ala Ala Asn Asn Tyr Ala Arg Gly Hi#s Tyr Thr Ile Gly Lys             100       #           105      #           110 Glu Ile Val Asp Leu Ala Leu Asp Arg Ile Ar#g Lys Leu Ala Asp Asn         115           #       120          #       125 Cys Thr Gly Leu Gln Gly Phe Met Val Phe Hi#s Ala Val Gly Gly Gly     130               #   135              #   140 Thr Gly Ser Gly Leu Gly Ala Leu Leu Leu Gl#u Arg Leu Ser Val Asp 145                 1 #50                 1#55                 1 #60 Tyr Gly Lys Lys Ser Lys Leu Gly Tyr Thr Va#l Tyr Pro Ser Pro Gln                 165   #               170  #               175 Val Ser Thr Ala Val Val Glu Pro Tyr Asn Cy#s Val Leu Ser Thr His             180       #           185      #           190 Ser Leu Leu Glu His Thr Asp Val Ala Thr Me#t Leu Asp Asn Glu Ala         195           #       200          #       205 Ile Tyr Asp Leu Thr Arg Arg Ser Leu Asp Il#e Glu Arg Pro Ser Tyr     210               #   215              #   220 Thr Asn Val Asn Arg Leu Ile Gly Gln Val Va#l Ser Ser Leu Thr Ala 225                 2 #30                 2#35                 2 #40 Ser Leu Arg Phe Asp Gly Ala Leu Asn Val As#p Leu Thr Glu Phe Gln                 245   #               250  #               255 Thr Asn Leu Val Pro Tyr Pro Arg Ile His Ph#e Val Leu Thr Ser Tyr             260       #           265      #           270 Ala Pro Val Val Ser Ala Glu Lys Ala Tyr Hi#s Glu Gln Leu Ser Val         275           #       280          #       285 Ala Asp Ile Thr Asn Ser Val Phe Glu Pro Al#a Gly Met Leu Thr Lys     290               #   295              #   300 Cys Asp Pro Arg His Gly Lys Tyr Met Ser Cy#s Cys Leu Met Tyr Arg 305                 3 #10                 3#15                 3 #20 Gly Asp Val Val Pro Lys Asp Val Asn Ala Al#a Ile Ala Thr Ile Lys                 325   #               330  #               335 Thr Lys Arg Thr Ile Gln Phe Val Asp Trp Cy#s Pro Thr Gly Phe Lys             340       #           345      #           350 Cys Gly Ile Asn Tyr Gln Pro Pro Thr Val Va#l Pro Gly Gly Asp Leu         355           #       360          #       365 Ala Lys Val Gln Arg Ala Val Cys Met Ile Al#a Asn Ser Thr Ala Ile     370               #   375              #   380 Ala Glu Val Phe Ala Arg Ile Asp His Lys Ph#e Asp Leu Met Tyr Ser 385                 3 #90                 3#95                 4 #00 Lys Arg Ala Phe Val His Trp Tyr Val Gly Gl#u Gly Met Glu Glu Gly                 405   #               410  #               415 Glu Phe Ser Glu Ala Arg Glu Asp Leu Ala Al#a Leu Glu Lys Asp Tyr             420       #           425      #           430 Glu Glu Val Gly Ala Glu Ser Ala Asp Asp Me#t Gly Glu Glu Asp Val         435           #       440          #       445 Glu Glu Tyr     450 <210> SEQ ID NO 109 <211> LENGTH: 1908<212> TYPE: DNA <213> ORGANISM: Leishmania major <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(1908)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 109taacgctata taagtatcag tttctgtact ttattgtaag cgcaatcgag tt#tcaacagc     60taacaaaatg gtgaacttta ccgtcgatca ggtccgcgag ctgatggact at#ccggacca    120gatccggaac atgtccgtga ttgctcacgt cgaccacggc aagtcgacgc tg#tccgactc    180tctcgttggt gctgccggca tcatcaagat ggaggaggct ggcgataagc gg#atcatgga    240tacacgcgcg gatgagatcg cgcgtggtat cacgatcaag tccaccgcca tc#tccatgca    300ctaccacgtg ccgaaggaga tgatcggcga tctggatgac gacaagcgcg ac#ttcctgat    360caacctgatc gactcccccg gacacgtcga cttcagctcc gaggtgactg cc#gctcttcg    420tgtgacggac ggcgcgctgg tcgtggtgga ctgcgtggag ggcgtgtgcg tg#cagacgga    480gacggtgctg cgccaggcgc tgacggagcg catccgccct gttgtgttca tc#aacaaggt    540ggaccgcgcc atccttgagc tccaactgga ccccgaggag gcgtaccagg gc#ttcgtgaa    600gacgctgcag aacgtgaacg tggtggttgc cacgtacaat gaccccagca tg#ggcgacgt    660gcaggtgtcc cccgagaagg gcactgtggc gatcggctct ggcctgcagg cg#tgggcgtt    720ctcgctgacc cgctttgcga acatgtatgc ggcgaagttc ggcgtggacg ag#ctgaagat    780gcgcgagcgc ctgtggggcg acaacttctt tgacgcgaag aacaagaagt gg#atcaagca    840ggagacgaac gccgatggcg agcgcgtgcg ccgcgcgttc tgccagttct gc#ctagaccc    900catctaccag atcttcgacg ctgtgatgaa cgagaagaag gacaaggtgg ac#aagatgct    960caagtcgctg cacgtgacgc tgacggctga ggagcgcgag caggtgccga ag#aagcttct   1020gaagacggtg atgatgaagt tcctgccggc tgctgagacg ctgctacaga tg#atcgtggc   1080gcacctgccg tcgcccaaga aggcgcaggc gtaccgtgcg gagatgctgt ac#tctggcga   1140ggcgtcgccg gaggacaagt acttcatggg tatcaagaac tgcgaccccg ct#gcgccgct   1200catgctgtac atcagcaaga tggtgccgac ggccgaccgc ggccgcttct tc#gcctttgg   1260ccgcatcttc tccggtaagg tgcgcagcgg ccagaaggtg cgcatcatgg gt#aacaacta   1320cgtctacggc aagaagcagg acctgtacga ggacaagcct gtgcagcgct cc#gtgctgat   1380gatgggccgc taccaggagg ccgtggagga catgccgtgc ggtaacgtgg tg#ggccttgt   1440gggcgtggac aagtacatcg tgaagtccgc gacgatcacg gacgatggcg ag#agcccgca   1500cccgctgcgc gacatgaagt actctgtgtc gcccgtcgtg cgtgtggccg tg#gaggcgaa   1560gaacccgtcc gacctgccga agcttgtgga gggcctgaag cgccttgcca ag#tccgaccc   1620gctggtggtg tgcagcattg aggagtctgg cgagcacatt gttgccggcg ct#ggcgagct   1680tcaccttgag atttgcctga aggatctcca ggaggacttc atgaacggcg cg#ccgctnaa   1740gatctccgag ccggtggtgt cgttccgcga gacggtgacg gatgtgtcgt cg#cagcagtg   1800cctgtcgaag tctgcgaaca agcacaaccg tctcttctgc cgcggtgcgc cg#ctnacaga   1860 gganctggcg ctggcgatng angaaggcac cgctggtccc gangcgga  #              1908 <210> SEQ ID NO 110 <211> LENGTH: 845<212> TYPE: PRT <213> ORGANISM: Leishmania major <220> FEATURE:<221> NAME/KEY: VARIANT <222> LOCATION: (1)...(845)<223> OTHER INFORMATION: Xaa = Any Amino Aci #d <400> SEQUENCE: 110Met Val Asn Phe Thr Val Asp Gln Val Arg Gl #u Leu Met Asp Tyr Pro 1               5   #                10   #                15Asp Gln Ile Arg Asn Met Ser Val Ile Ala Hi #s Val Asp His Gly Lys            20       #            25       #            30Ser Thr Leu Ser Asp Ser Leu Val Gly Ala Al #a Gly Ile Ile Lys Met        35           #        40           #        45Glu Glu Ala Gly Asp Lys Arg Ile Met Asp Th #r Arg Ala Asp Glu Ile    50               #    55               #    60Ala Arg Gly Ile Thr Ile Lys Ser Thr Ala Il #e Ser Met His Tyr His65                   #70                   #75                   #80Val Pro Lys Glu Met Ile Gly Asp Leu Asp As #p Asp Lys Arg Asp Phe                85   #                90   #                95Leu Ile Asn Leu Ile Asp Ser Pro Gly His Va #l Asp Phe Ser Ser Glu            100       #           105       #           110Val Thr Ala Ala Leu Arg Val Thr Asp Gly Al #a Leu Val Val Val Asp        115           #       120           #       125Cys Val Glu Gly Val Cys Val Gln Thr Glu Th #r Val Leu Arg Gln Ala    130               #   135               #   140Leu Thr Glu Arg Ile Arg Pro Val Val Phe Il #e Asn Lys Val Asp Arg145                 1 #50                 1 #55                 1 #60Ala Ile Leu Glu Leu Gln Leu Asp Pro Glu Gl #u Ala Tyr Gln Gly Phe                165   #               170   #               175Val Lys Thr Leu Gln Asn Val Asn Val Val Va #l Ala Thr Tyr Asn Asp            180       #           185       #           190Pro Ser Met Gly Asp Val Gln Val Ser Pro Gl #u Lys Gly Thr Val Ala        195           #       200           #       205Ile Gly Ser Gly Leu Gln Ala Trp Ala Phe Se #r Leu Thr Arg Phe Ala    210               #   215               #   220Asn Met Tyr Ala Ala Lys Phe Gly Val Asp Gl #u Leu Lys Met Arg Glu225                 2 #30                 2 #35                 2 #40Arg Leu Trp Gly Asp Asn Phe Phe Asp Ala Ly #s Asn Lys Lys Trp Ile                245   #               250   #               255Lys Gln Glu Thr Asn Ala Asp Gly Glu Arg Va #l Arg Arg Ala Phe Cys            260       #           265       #           270Gln Phe Cys Leu Asp Pro Ile Tyr Gln Ile Ph #e Asp Ala Val Met Asn        275           #       280           #       285Glu Lys Lys Asp Lys Val Asp Lys Met Leu Ly #s Ser Leu His Val Thr    290               #   295               #   300Leu Thr Ala Glu Glu Arg Glu Gln Val Pro Xa #a Lys Leu Leu Lys Thr305                 3 #10                 3 #15                 3 #20Val Met Met Xaa Phe Leu Pro Ala Ala Glu Th #r Leu Leu Gln Met Ile                325   #               330   #               335Val Ala His Leu Pro Ser Pro Lys Lys Ala Gl #n Ala Tyr Arg Ala Glu            340       #           345       #           350Met Leu Tyr Ser Gly Glu Ala Ser Pro Glu As #p Lys Tyr Phe Met Gly        355           #       360           #       365Ile Lys Asn Cys Asp Pro Ala Ala Pro Leu Me #t Leu Tyr Ile Ser Lys    370               #   375               #   380Met Val Pro Thr Ala Asp Arg Gly Arg Phe Ph #e Ala Phe Gly Arg Ile385                 3 #90                 3 #95                 4 #00Phe Ser Gly Lys Val Arg Ser Gly Gln Lys Va #l Arg Ile Met Gly Asn                405   #               410   #               415Asn Tyr Val Tyr Gly Lys Lys Gln Asp Leu Ty #r Glu Asp Lys Pro Val            420       #           425       #           430Gln Arg Ser Val Leu Met Met Gly Arg Tyr Gl #n Glu Ala Val Glu Asp        435           #       440           #       445Met Pro Cys Gly Asn Val Val Gly Leu Val Gl #y Val Asp Lys Tyr Ile    450               #   455               #   460Val Lys Ser Ala Thr Ile Thr Asp Asp Gly Gl #u Ser Pro His Pro Leu465                 4 #70                 4 #75                 4 #80Arg Asp Met Lys Tyr Ser Val Ser Pro Val Va #l Arg Val Ala Val Glu                485   #               490   #               495Ala Lys Asn Pro Ser Asp Leu Pro Lys Leu Va #l Glu Gly Leu Lys Arg            500       #           505       #           510Leu Ala Lys Ser Asp Pro Leu Val Val Cys Se #r Ile Glu Glu Ser Gly        515           #       520           #       525Glu His Ile Val Ala Gly Ala Gly Glu Leu Hi #s Leu Glu Ile Cys Leu    530               #   535               #   540Lys Asp Leu Gln Glu Asp Phe Met Asn Gly Al #a Pro Leu Lys Ile Ser545                 5 #50                 5 #55                 5 #60Glu Pro Val Val Ser Phe Arg Glu Thr Val Th #r Asp Val Ser Ser Gln                565   #               570   #               575Gln Cys Leu Ser Lys Ser Ala Asn Lys His As #n Arg Leu Phe Cys Arg            580       #           585       #           590Gly Ala Pro Leu Thr Glu Glu Leu Ala Leu Al #a Met Glu Glu Gly Thr        595           #       600           #       605Ala Gly Pro Glu Ala Asp Pro Lys Val Arg Al #a Arg Phe Leu Ala Asp    610               #   615               #   620Asn Tyr Glu Trp Asp Val Gln Glu Ala Arg Ly #s Ile Trp Cys Tyr Gly625                 6 #30                 6 #35                 6 #40Pro Asp Asn Arg Gly Pro Asn Val Val Val As #p Val Thr Lys Gly Val                645   #               650   #               655Gln Asn Met Ala Glu Met Lys Asp Ser Phe Va #l Ala Ala Trp Gln Trp            660       #           665       #           670Ala Thr Arg Glu Gly Val Leu Cys Asp Glu As #n Met Arg Gly Val Arg        675           #       680           #       685Val Asn Val Glu Asp Val Thr Met His Ala As #p Ala Ile His Arg Gly    690               #   695               #   700Gly Val Gln Ile Ile Pro Thr Ala Arg Arg Va #l Phe Tyr Ala Cys Cys705                 7 #10                 7 #15                 7 #20Leu Thr Ala Ser Pro Arg Leu Met Glu Pro Me #t Phe Val Val Asp Ile                725   #               730   #               735Gln Thr Val Glu His Ala Met Gly Gly Ile Ty #r Gly Val Leu Thr Arg            740       #           745       #           750Arg Arg Gly Val Ile Ile Gly Glu Glu Asn Ar #g Pro Gly Thr Pro Ile        755           #       760           #       765Tyr Asn Val Arg Ala Tyr Leu Pro Val Ala Gl #u Ser Phe Gly Phe Thr    770               #   775               #   780Ala Asp Leu Arg Ala Gly Thr Gly Gly Gln Al #a Phe Pro Gln Cys Val785                 7 #90                 7 #95                 8 #00Phe Asp His Trp Gln Glu Tyr Pro Gly Asp Pr #o Leu Glu Pro Lys Ser                805   #               810   #               815Leu Ala Asn Thr Thr Thr Leu Gly Ile Arg Th #r Arg Lys Gly Leu Lys            820       #           825       #           830Pro Asp Ile Pro Gly Leu Asp Gln Phe Met As #p Lys Leu        835           #       840           #       845<210> SEQ ID NO 111 <211> LENGTH: 997 <212> TYPE: DNA<213> ORGANISM: Leishmania major <400> SEQUENCE: 111ggatccgccg ccaccatggt gaacgtgtgc gttgttggtg ctgccggcgg ca#ttggccag     60tcgctgtcgc tgctgttggt gcgccagctg ccgtacggga gcacgttgtc gc#tgttcgac    120gttgtgggcg ctgcaggcgt cgcagcagac ctgtcgcatg tggacaacgc cg#gtgtgcag    180gtgaagtttg cggagggcaa gatcggccat aagcgcgacc ctgcgctggc ag#agcttgcg    240aagggcgtgg atgtgtttgt aatggtggct ggcgttccac gcaagccggg ca#tgacgcgc    300gacgaccttt tcaaaatcaa cgccggaatc atcctggacc ttgtgctgac gt#gcgcgtcg    360tcgagtccaa aggcggtgtt ctgcattgtg acgaaccctg tgaacagcac gg#tcgcgatc    420gcggcagagg cgctgaagag ccttggcgta tacgaccgaa accggctgct tg#gcgtgtcg    480ctgctggacg ggctgcgcgc gacgtgcttc atcaacgagg cgcgcaagcc ct#tagtcgtg    540tcgcaggtac cagttgttgg cgggcacagc gacacaacga ttgtgccgtt gt#tctaccag    600ctaccggggc cgttgccgga gcaggcgacg ctggacaaga tcgtgaagcg cg#tgcaggtc    660gcaggcacag aagtggtgaa ggcgaaggcc gggcgcgggt ctgcgacgct gt#cgatggcg    720gaggctggcg cgcggttcgc gttgaaggtt gtggagggtc tgaccggcac gg#gtaacccg    780ctggtgtacg catatgtaga cacagacggg cagcacgaga cgacgttcct cg#cgatccct    840gtggtgcttg gcatgaatgg aatcgagaag cgcctgccga ttggtccgct gc#actcgacg    900gaggaaacgc tgctgaaggc ggcactgccg gtgatcaaga agaatatcgt ga#agggcagc    960 gagttcgcgc gctcacacct gtagcacctc agaattc      #                   #     997 <210> SEQ ID NO 112 <211> LENGTH: 322<212> TYPE: PRT <213> ORGANISM: Leishmania major <400> SEQUENCE: 112Met Val Asn Val Cys Val Val Gly Ala Ala Gl #y Gly Ile Gly Gln Ser 1               5   #                10   #                15Leu Ser Leu Leu Leu Val Arg Gln Leu Pro Ty #r Gly Ser Thr Leu Ser            20       #            25       #            30Leu Phe Asp Val Val Gly Ala Ala Gly Val Al #a Ala Asp Leu Ser His        35           #        40           #        45Val Asp Asn Ala Gly Val Gln Val Lys Phe Al #a Glu Gly Lys Ile Gly    50               #    55               #    60His Lys Arg Asp Pro Ala Leu Ala Glu Leu Al #a Lys Gly Val Asp Val65                   #70                   #75                   #80Phe Val Met Val Ala Gly Val Pro Arg Lys Pr #o Gly Met Thr Arg Asp                85   #                90   #                95Asp Leu Phe Lys Ile Asn Ala Gly Ile Ile Le #u Asp Leu Val Leu Thr            100       #           105       #           110Cys Ala Ser Ser Ser Pro Lys Ala Val Phe Cy #s Ile Val Thr Asn Pro        115           #       120           #       125Val Asn Ser Thr Val Ala Ile Ala Ala Glu Al #a Leu Lys Ser Leu Gly    130               #   135               #   140Val Tyr Asp Arg Asn Arg Leu Leu Gly Val Se #r Leu Leu Asp Gly Leu145                 1 #50                 1 #55                 1 #60Arg Ala Thr Cys Phe Ile Asn Glu Ala Arg Ly #s Pro Leu Val Val Ser                165   #               170   #               175Gln Val Pro Val Val Gly Gly His Ser Asp Th #r Thr Ile Val Pro Leu            180       #           185       #           190Phe Tyr Gln Leu Pro Gly Pro Leu Pro Glu Gl #n Ala Thr Leu Asp Lys        195           #       200           #       205Ile Val Lys Arg Val Gln Val Ala Gly Thr Gl #u Val Val Lys Ala Lys    210               #   215               #   220Ala Gly Arg Gly Ser Ala Thr Leu Ser Met Al #a Glu Ala Gly Ala Arg225                 2 #30                 2 #35                 2 #40Phe Ala Leu Lys Val Val Glu Gly Leu Thr Gl #y Thr Gly Asn Pro Leu                245   #               250   #               255Val Tyr Ala Tyr Val Asp Thr Asp Gly Gln Hi #s Glu Thr Thr Phe Leu            260       #           265       #           270Ala Ile Pro Val Val Leu Gly Met Asn Gly Il #e Glu Lys Arg Leu Pro        275           #       280           #       285Ile Gly Pro Leu His Ser Thr Glu Glu Thr Le #u Leu Lys Ala Ala Leu    290               #   295               #   300Pro Val Ile Lys Lys Asn Ile Val Lys Gly Se #r Glu Phe Ala Arg Ser305                 3 #10                 3 #15                 3 #20His Leu <210> SEQ ID NO 113 <211> LENGTH: 1617 <212> TYPE: DNA<213> ORGANISM: Leishmania major and chagasi <400> SEQUENCE: 113cggaatacgt acctcctccc ccttcttggt agaagaacaa caacaacgtt ca#agacgacg     60ccgcgccttc ttgtaccgca tttgcttctg agcacgttca atccgtgcct tg#caaacatg    120gaggcgtaca agaagctgga aacgatcttt acgaaggtct accgcctgga cc#acttcctc    180ggtctgggca actgggacat gaacacaaac atgcccccca agggcgagga at#cacgcggt    240gaggcgatgg cgatgctctc ggagctccgc tttggcttca tcacggcacc gg#aggtgaaa    300agcctgattg agagtgccac caagggcagc gaggagctga atgcggtgca gc#gcgctaac    360ttgcgggaga tgaggcgtgc gtggaagagc gccaccgcct tgccggctga gt#ttgtgggc    420cgcaagatgc gcctcacgac acacgcgcac agcgtgtggc gcgacagccg ca#aagcaaat    480gacttcgcca agttcctacc ggtgctcagg gacctggtgg cgctcgcccg tg#aggagggc    540tcatacctcg ccgccggcac ctccctctcc ccgtatgagg cgctcatgaa cg#agtacgag    600ccaggaatca cgacacaaaa gctggatgag gtgtacgcaa atgtaaagtc gt#ggctgccg    660cagctgctaa aggacattgt gcagaagcag tccggcgagt cggtgattgc gt#tctcgcat    720aagttcccgc aggacaagca ggaagcactg tgcaaggaat tcatgaagat ct#ggcacttc    780gacaccgatg ccggtcgcct cgacgtcagc ccccaccctt tcacgggaat ga#cgaaggag    840gactgccgac tcacaacaaa ctacatcgaa gacacgtttg ttcagagctt gt#atggcgtc    900atccacgaga gtgggcatgg caagtacgag cagaactgtg gcccacgcga gc#acatcacg    960cagccggtgt gcaacgcccg ctctcttggc ctgcatgaga gccagagcct ct#ttgcggag   1020tttcagatcg gccacgcgac gcccttcatc gactacctca caactcgcct tc#ctgagttc   1080ttcgaggcgc agccagcgtt ctcgcaggac aacatgcgca agtcgctgca gc#aggtgaag   1140ccgggctaca ttcgcgtcga tgccgatgag gtgtgctacc ctctgcacgt ga#tcctgcgc   1200tacgagatcg agcgcgactt gatggagggc aaaatggagg tggaagacgt gc#cgcgcgcg   1260tggaacgcaa agatgcagga gtacttgggt ctctcaacgg agggccgtga cg#acgttggg   1320tgcctgcagg acgtgcattg gtccatgggt gcgctcggct actttccgac gt#actcgctc   1380ggcgccatgt atgcggcgca gatcatggcg agcatccgaa aggagctggg ag#acgacaag   1440gtggatgagt gcctgcgcac cggtgagctc ggccccctcc tggaaaagca gc#aggagaag   1500atctgggatc atgggtgcct gtacgagacg gacgacctca tgacgcgtgc ga#cgggcgag   1560acgctgaacc ccgagtacct gcgccgccac ctggaggcgc gctacctaaa cg#cctga      1617 <210> SEQ ID NO 114 <211> LENGTH: 582 <212> TYPE: DNA<213> ORGANISM: Leishmania major and chagasi <400> SEQUENCE: 114atgcatcacc atcaccatca cgatcccccg ggctgcagga attcggcacg ag#agctgcag     60cagcgcctag acaccgccac gcagcagcgc gccgagctgg aggcacgggt gg#cacggctg    120gccgcggacc gcgacgaggc gcgccagcag ctggccgcga acgccgagga gc#tgcagcag    180cgcctagaca ccgccacgca gcagcgcgcc gagctggagg cacgggtggc ac#ggctggcc    240gcggacggcg acgaggcccg ccagcagctg gccgcgaacg ccgaggagct gc#agcagcgc    300ctagacaccg ccacgcagca gcgcgccgag ctggaggcac aggtggcacg gc#tggccgcg    360aacgccgagg agctgcagca gcgcctagac accgccacgc agcagcgcgc cg#agctggag    420gcacgggtgg cacggctggc cgcggaccgc gacgaggcgc gccagcagct gg#ccgcgaac    480gccgaggagc tgcagcagcg cctagacacc gccacgcagc agcgcgccga gc#tggaggca    540 caggtggcac ggctggccgc gaacgccgct cgtgccgaat aa    #                   # 582 <210> SEQ ID NO 115 <211> LENGTH: 7065<212> TYPE: DNA <213> ORGANISM: Leishmania major and chagasi<400> SEQUENCE: 115atgtccactc ctgtcagtgg agtagtcccg caggaccgct ggcagcctca gc#agagggtg     60aaggtctgtc agtaccagga ctgcggcgcc cccttcggct tcttctccac ca#aggtcaac    120tgccaccgct gcggcatcgt cctctgcagc aagtgcgccg ccaccaagac cg#tgattccc    180cgctactaca gcaatgaaac cgttcccgtc tgccagcgct gctaccaggt gg#tggagcgc    240tacaaggaac gcgggtcagt gacaccggga tatgttgttc actctaccac ca#tcagcgcc    300acgccggctc gatcctcgcc ggtgccgcca cttcacacga ctccagccct cc#ggccgcat    360gcaccctcgc cgcagcccgc ttccgtcgtg tccacggcaa cactcgtcca tc#ccgtagaa    420gaagacgcag tgtccacaaa accctccgtc agcgaggccg acctgcacgc cc#tgcgaagc    480atcatcgaga ccctccagca agcactcaac gatgaacaac acaacgccgc cc#tagccgcc    540acgtccgctg ccgagcagct ccgaacagcc aaagaagaga acacagcact ca#aaagcacc    600gcacatctcc tgcagcagcg cctagacacc gccacgcagc agcgcgccga gc#tggaggca    660cgggtggcac ggctggccgc ggaccgcgac gaggcgcgcc agcagctggc cg#cgaacgcc    720gaggagctgc agcagcgcct agacaccgcc acgcagcagc gcgccgagct gg#aggcacgg    780gtggcacggc tggccgcgga ccgcgacgag gcgcgccagc agctggccgc ga#acgccgag    840gagctgcagc agcgcctaga caccgccacg cagcagcgcg ccgagctgga gg#cacaggtg    900gcacggctgg ccgcggacgg cgacgaggcg cgccagcagc tggccgcgaa cg#ccgaggag    960ctgcagcagc gcctagacac cgccacgcag cagcgcgccg agctggaggc ac#gggtggca   1020cggctggccg cggaccgcga cgaggcgcgc cagcagctgg ccgcgaacgc cg#aggagctg   1080cagcagcgcc tagacaccgc cacgcagcag cgcgccgagc tggaggcaca gt#tggcacgg   1140ctggccgcgg accgcgacga ggcgcgccag cagctggccg cgaacgccga gg#agctgcag   1200cagcgcctag acaccgccac gcagcagcgc gccgagctgg aggcacaggt gg#cacggctg   1260gccgcgaacg ccgaggagct gcagcagcgc ctagacaccg ccacgcagca gc#gcgccgag   1320ctggaggcac gggtggcacg gctggccgcg gaccgcgacg aggcgcgcca gc#agctggcc   1380gcgaacgccg aggagctgca gcagcgccta gacaccgcca cgcagcagcg cg#ccgagctg   1440gaggcacggg tggcacggct ggccgcggac ggcgacgagg cccgccagca gc#tggccgcg   1500aacgccgagg agctgcagca gcgcctagac accgccacgc agcagcgcgc cg#agctggag   1560gcacaggtgg cacggctggc cgcgaacgcc gaggagctgc agcagcgcct ag#acaccgcc   1620acgcagcagc gcgccgagct ggaggcacgg gtggcacggc tggccgcgga cc#gcgacgag   1680gcgcgccagc agctggccgc gaacgccgag gagctgcagc agcgcctaga ca#ccgccacg   1740cagcagcgcg ccgagctgga ggcacaggtg gcacggctgg ccgcgaacgc cg#aggagctg   1800cagcagcgcc tagacaccgc cacgcagcag cgcgccgagc tggaggcacg gg#tggcacgg   1860ctggccgtgg accgcgacga ggcgcgccag cagctggccg cgaacgccga gg#agctgcag   1920cagcgcctag acaccgccac gcagcagcgc gccgagctgg aggcacaggt gg#cacggctg   1980gccgcggacc gcgacgaggc gcgccagcag ctggccgcga acgccgagga gc#tgcagcag   2040cgcctagaca ccgccacgca gcagcgcgcc gagctggagg cacagttggc ac#ggctggcc   2100gcggaccgcg acgaggcgcg ccagcagctg gccgcgaacg ccgaggagct gc#agcagcgc   2160ctagacaccg ccacgcagca gcgcgccgag ctggaggcac aggtggcacg gc#tggccgcg   2220gaccgcgacg aggcgcgcca gcagctggcc gcgaacgccg aggagctgca gc#agcgccta   2280gacaccgcca cgcagcagcg cgccgagctg gaggcacagt tggcacggct gg#ccgcggac   2340cgcgacgagg cgcgccagca gctggccgcg aacgccgagg agctgcagca gc#gcctagac   2400accgccacgc agcagcgcgc cgagctggag gcacaggtgg cacggctggc cg#cggaccgc   2460gacgaggcgc gccagcagct ggccgcgaac gccgaggagc tgcagcagcg cc#tagacacc   2520gccacgcagc agcgcgccga gctggaggca caggtggcac ggctggccgc gg#accgcgac   2580gaggcccgcc agcagctggc cgcgaacgcc gaggagctgc agcagcgcct ag#acaccgcc   2640acgcagcagc gcgccgagct ggaggcacag gtggcacggc tggccgcgaa cg#ccgaggag   2700ctgcagcagc gcctagacac cgccacgcag cagcgcgccg agctggaggc ac#gggtggca   2760cggctggccg cggaccgcga cgaggcgcgc cagcagctgg ccgcgaacgc cg#aggagctg   2820cagcagcgcc tagacaccgc cacgcagcag cgcgccgagc tggaggcaca gt#tggcacgg   2880ctggccgcgg accgcgacga ggcgcgccag cagctggccg cgaacgccga gg#agctgcag   2940cagcgcctag acaccgccac gcagcagcgc gccgagctgg aggcacagtt gg#cacggctg   3000gccgcggacc gcgacgaggc gcgccagcag ctggccgcga acgccgagga gc#tgcagcag   3060cgcctagaca ccgccacgca gcagcgcgcc gagctggagg cacaggtggc ac#ggctggcc   3120gcggaccgcg acgaggcgcg ccagcagctg gccgcgaacg ccgaggagct gc#agcagcgc   3180ctagacaccg ccacgcagca gcgcgccgag ctggaggcac gggtggcacg gc#tggccgcg   3240gaccgcgacg aggcgcgcca gcagctggcc gcgaacgccg aggagctgca gc#agcgccta   3300gacaccgcca cgcagcagcg cgccgagctg gaggcacagg tggcacggct gg#ccgcggac   3360ggcgacgagg cgcgccagca gctggccgcg aacgccgagg agctgcagca gc#gcctagac   3420accgccacgc agcagcgcgc cgagctggag gcacgggtgg cacggctggc cg#cggaccgc   3480gacgaggcgc gccagcagct ggccgcgaac gccgaggagc tgcagcagcg cc#tagacacc   3540gccacgcagc agcgcgccga gctggaggca cagttggcac ggctggccgc gg#accgcgac   3600gaggcgcgcc agcagctggc cgcgaacgcc gaggagctgc agcagcgcct ag#acaccgcc   3660acgcagcagc gcgccgagct ggaggcacag gtggcacggc tggccgcgga cg#gcgacgag   3720gcgcgccagc agctggccgc gaacgccgag gagctgcagc agcgcctaga ca#ccgccacg   3780cagcagcgcg ccgagctgga ggcacagttg gcacggctgg ccgcggaccg cg#acgaggcg   3840cgccagcagc tggccgcgaa cgccgaggag ctgcagcagc gcctagacac cg#ccacgcag   3900cagcgcgccg agctggaggc acaggtggca cggctggccg cgaacgccga gg#agctgcag   3960cagcgcctag acaccgccac gcagcagcgc gccgagctgg aggcacgggt gg#cacggctg   4020gccgcggacc gcgacgaggc gcgccagcag ctggccgcga acgccgagga gc#tgcagcag   4080cgcctagaca ccgccacgca gcagcgcgcc gagctggagg cacgggtggc ac#ggctggcc   4140gcggaccgcg acgaggcgcg ccagcagctg gccgcgaacg ccgaggagct gc#agcagcgc   4200ctagacaccg ccacgcagca gcgcgccgag ctggaggcac aggtggcacg gc#tggccgcg   4260aacgccgagg agctgcagca gcgcctagac accgccacgc agcagcgcgc cg#agctggag   4320gcacgggtgg cacggctggc cgcggaccgc gacgaggcgc gccagcagct gg#ccgcgaac   4380gccgaggagc tgcagcagcg cctagacacc gccacgcagc agcgcgccga gc#tggaggca   4440caggtggcac ggctggccgc ggaccgcgac gaggcgcgcc agcagctggc cg#cgaacgcc   4500gaggagctgc agcagcgcct agacaccgcc acgcagcagc gcgccgagct gg#aggcacgg   4560gtggcacggc tggccgcgga cggcgacgag gcccgccagc agctggccgc ga#acgccgag   4620gagctgcagc agcgcctaga caccgccacg cagcagcgcg ccgagctgga gg#cacagttg   4680gcacggctgg ccgcggaccg cgacgaggcg cgccagcagc tggccgcgaa cg#ccgaggag   4740ctgcagcagc gcctagacac cgccacgcag cagcgcgccg agctggaggc ac#gggtggca   4800cggctggccg cggacggcga cgaggcgcgc cagcagctgg ccgcgaacgc cg#aggagctg   4860cagcagcgcc tagacaccgc cacgcagcag cgcgccgagc tggaggcacg gg#tggcacgg   4920ctggccgcgg accgcgacga ggcgcgccag cagctggccg cgaacgccga gg#agctgcag   4980cagcgcctag acaccgccac gcagcagcgc gccgagctgg aggcacagtt gg#cacggctg   5040gccgcggacc gcgacgaggc gcgccagcag ctggccgcga acgccgagga gc#tgcagcag   5100cgcctagaca ccgccacgca gcagcgcgcc gagctggagg cacagttggc ac#ggctggcc   5160gcggacggcg acgaggcgcg ccagcagctg gccgcgaacg ccgaggagct gc#agcagcgc   5220ctagacaccg ccacgcagca gcgcgccgag ctggaggtgg agatggcagt tc#tcctgcgt   5280gagagggagg aagctcgcgg agagacagca gtggctggag agcaggtgca gc#tgtaccgg   5340gaaacggtcg aggaggagga gtgtttaaag gaggaacgat ggtgcctcga gt#cacgggtg   5400gcgcagctga gggaggcgtc cgcggccgcg aagcagcagc ggcaagaagt gg#cggcgaag   5460gcgaatgagg tgcaggagcg ccttgattcg atggcgcgtc ggtgcattgc gc#atgaggga   5520gatgcgccgc agcgggctga cgggagagac gacgccttgc ggcaacttgc ta#atctgcgt   5580gaagaggtga agctcagtga gaagcagaag gcgatggagc gtgttattcc gg#gtgttaga   5640gagcggcaga tgcgcctcga agcagctgag gagcagcggg cggacttgga ag#cacgcctc   5700gtcgacgagg ccggtgacct gcgctctcgt cctgctgcga gcacaaacga ag#tgaatctg   5760taccgcgacc tcgctctgca ggaacacgaa gcagcccaga atcggtgcac aa#cactggaa   5820gcgcaggtgg caagcctcac gagcgaccgc gacaacgggc gccagcaaga gt#cggctgac   5880ctcagcgagg cgcagcgtca cctcgacaac gtgcaggagc gcgacatggc cc#atcatcgg   5940tgtgccgcgt tggaggagca gaatgctgct atggcctcgg agttgcaagc gg#tcaaggct   6000aaactgcggc aggcctcagt gaaggcctcc tcgttgatga cgcgactctc tg#catcatcc   6060tcgggagctg gtggtgtctc tgcccgtgtg agagtgggag gcagctctgc cg#tgccacaa   6120gcggcgccac accgggatgc ggagctcatc gccgaggtgg gcgaacgcct tc#gcgaaagg   6180ggggaggcca tgcgactgtt ggccgaaggc gtcgaactgc gcgagcgcgc gc#ggccgctg   6240gaacgggtgc tcgccgagaa gctgatcggc gaccgtcgca cgagcgacgc gg#aagaggtc   6300gccacggagc ccacgcaggt gcggcggaac gctgcgcact cgcggcacct gg#actcgcgt   6360gaggcgcagc tggacgagcg cgcggcacgg ctgagggaaa aggagcagca gc#tgctgcgc   6420gtcgctcgtg aactgcagac taagtcgcgc gcccttcaag tattatacgc ac#gggcgctg   6480aataggccac aggtgacgtc gttgctgctg acggccgacg gcgacgatac ct#cctacccc   6540gacacaccac agcaacagca gcaaggcaca cgcacgccgc tcagagaacc tg#tatattcg   6600ctggacagcg aggtggcgca ctacggccga actgcagggg ccgcagtgag ca#gtggtctc   6660gcatcacctc tgccgaggga gccgccacgg gctcggatgg tgcaccgcgc tg#tggaggca   6720acgggcacag aggaagacac gcaggtgcgc ctcacggctg caacggaggc tt#accgcgac   6780gtactgtacg agcacattct tgagtcgaat gggctccaag gggtggatgt gt#tggctcag   6840tacctgcccc accacacctc cggcggtggg ctgaagacac cgcgactgcc gg#ggagcggc   6900attatatcga agacccgggc aatgctacga gcgctggagg agcgcctggg tg#cttcccgt   6960ggcgtcgggc gtggagtcga cccggcggtg caggagcgaa gcctggaagc gt#tccggcgg   7020 ctcgaggccg ccttgtctgc tctctgtgga ggcagccatg cgtag   #                7065 <210> SEQ ID NO 116 <211> LENGTH: 2589<212> TYPE: DNA <213> ORGANISM: Leishmania major and chagasi<220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)...(2589)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 116gtmccttcca agcttgcaat ccgtctcatg cgcagctctc gcggcggctc tc#tcgcggtg     60gtcgccttgg cggtttgcct ggcagtgctc gccgcgatcg gcacgtgtgt tt#tcgactcg    120caagagatcg gcggcagctc cttcaccttc gtaggctggt cgtctgccag ca#aggaggaa    180tcctaccagg gctgcaccct gaccgggaag gcattcagaa ttcaaggcgc tg#caagctct    240ctgtcggacg acgccacgct gcccggtggg atcctgcggt tctccagcct gc#ttgtatcg    300aacggctaca tcgtcgtgga taagtacttt ccccggaaca ccaacatcac ca#tcaaagat    360gccagcggca ccgtcgctgc ggggatgcct ttcatcgacg ccaacacggc ga#tatactct    420gatcagctca gcatcgtggt caccgactcc actctcagtt gggctgccgc ac#ggtcgggt    480cagagcatgg tgcgtgcacc cttcactatt cagctctcca gctctctctt cg#tgctcggc    540agcaccgtga cgcaggcctc gtcagtggta gaggtggtgg gcccgagcag ca#tctcccag    600aagtctgcat tggctgtgga ctacgccaag tgcaccggct gtgctcaggg tc#tggtgcac    660ttcaccgact ttgtcagagt gtgggaccgc agcttgcttc gcgtctccca ct#ccagcgtc    720aagggcgcca ccggcaagcc gctgatcggc atagcacaga gcgcaggggc ga#gcctggcg    780gtggaaaaca gtctgttcgt tgtcgagaac gtgtcatcgc ccaccagtaa cc#tcatcgac    840gcggccgtca gaatgggcac cgatgcgcag atcaccctac gcgctgtgac gg#tgaagagc    900attggtgcaa cgatggccgg cagcgtcact gcgcagctct tgacggcaga cg#acatcgcc    960caacaaattc cctccatttc cgtggtgcca gacacccgct gcgctgcggc ct#gcgtcccc   1020acggccacgg tcgactccag ctgcaagtgt gcctgcaatg ccgacatgcc ga#acatgaac   1080ttctgcacgg ccatgaagga cccctacaca aactatgcct acctcggctg ct#cggcgggc   1140tgcacaacgt gcttcaacga gaccgcctgc ctcgagtgca ggccgagcta cg#agatgctg   1200cccgacatga catgctcgct gaccggactt cagtgcacag acccgaactg ca#agacctgc   1260acaacttacg gtcagtgcac agactgcaac gacggctacg gtctcacctc ct#ccagcgtt   1320tgcgtgcgct gcagtgtagc gggctgcaag agctgccccg tcgacgctaa cg#tctgcaaa   1380gtgtgtctcg gcggcagcga gccgatcaac aatatgtgcc cctgcaccga cc#ccaactgc   1440gccagctgcc ccagcgacgc tggcacgtgc actcagtgcg cgaacggcta cg#gtctcgtg   1500gacggcgcct gtgtgagatg ccaggagccc aactgcttca gctgcgacag cg#acgcgaat   1560aagtgcacac aatgtgcgcc gaactactac ctcaccccgc tcttgacctg ct#ccccggtg   1620gcctgcaaca tcgagcactg catgcagtgc gacccacaga cgccgtcgcg ct#gccaggag   1680tgcgtgtccc cctacgtggt tgacagctac gacggcctct gcaggctctc cg#atgcctgc   1740tccgtgccca actgcaagaa gtgcgagacc ggtacctcca ggctctgcgc cg#agtgcgac   1800accggctaca gtctctccgc cgacgcgacg agctgcagca gtccaaccac gc#agccgtgc   1860gaggtggagc actgcaacac atgtgtgaac ggcgatagca cccgctgtgc ct#actgcaac   1920accggctact acgtctccga tggcaagtgc aaggccatgc agggctgcta cg#tgtcgaac   1980tgcgcgcagt gcatgctgct tgacagcacc aagtgctcca cgtgcgtgaa ag#ggtacctg   2040ctcacgtcgt cctacagttg cgtctcgcag aaagtcatca acagtgcggc cg#cgccctac   2100tctctgtggg tggccgccgc cgtgctcctc acctcttttg ccatgcacct ag#cataggct   2160agcccgggac gcgtggatcc tcgcaatccc taggaggatt aggcaagggc tt#gagctcac   2220gctcttgtga gggacagaaa tacaatcagg ggcagtatat gaatactcca tg#gagaaacc   2280cagatctacg tatgatcagc ctcgactgtg ccttctagtt gccagccatc tg#ttgtttgc   2340ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct tt#cctaataa   2400aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gg#gtggggtg   2460gggcangaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg gg#atgcggtg   2520ggctctatgg cttctgaggc ggaaagaacc agctggggct cgacagctcg ag#ctaggacc   2580 gctatcang                 #                  #                   #       2589 <210> SEQ ID NO 117 <211> LENGTH: 909<212> TYPE: DNA <213> ORGANISM: Leishmania major and chagasi<400> SEQUENCE: 117atgcatcacc atcaccatca cgactcgcaa gagatcggcg gcagctcctt ca#ccttcgta     60ggctggtcgt ctgccagcaa ggaggaatcc taccagggct gcaccctgac cg#ggaaggca    120ttcagaattc aaggcgctgc aagctctctg tcggacgacg ccacgctgcc cg#gtgggatc    180ctgcggttct ccagcctgct tgtatcgaac ggctacatcg tcgtggataa gt#actttccc    240cggaacacca acatcaccat caaagatgcc agcggcaccg tcgctgcggg ga#tgcctttc    300atcgacgcca acacggcgat atactctgat cagctcagca tcgtggtcac cg#actccact    360ctcagttggg ctgccgcacg gtcgggtcag agcatggtgc gtgcaccctt ca#ctattcag    420ctctccagct ctctcttcgt gctcggcagc accgtgacgc aggcctcgtc ag#tggtagag    480gtggtgggcc cgagcagcat ctcccagaag tctgcattgg ctgtggacta cg#ccaagtgc    540accggctgtg ctcagggtct ggtgcacttc accgactttg tcagagtgtg gg#accgcagc    600ttgcttcgcg tctcccactc cagcgtcaag ggcgccaccg gcaagccgct ga#tcggcata    660gcacagagcg caggggcgag cctggcggtg gaaaacagtc tgttcgttgt cg#agaacgtg    720tcatcgccca ccagtaacct catcgacgcg gccgtcagaa tgggcaccga tg#cgcagatc    780accctacgcg ctgtgacggt gaagagcatt ggtgcaacga tggccggcag cg#tcactgcg    840cagctcttga cggcagacga catcgcccaa caaattccct ccatttccgt gg#tgccagac    900 acccgctaa                 #                  #                   #        909 <210> SEQ ID NO 118 <211> LENGTH: 538<212> TYPE: PRT <213> ORGANISM: Leishmania major and chagasi<400> SEQUENCE: 118 Arg Asn Thr Tyr Leu Leu Pro Leu Leu Gly Ar#g Arg Thr Thr Thr Thr                    #5                  #10                   #15 Phe Lys Thr Thr Pro Arg Leu Leu Val Pro Hi#s Leu Leu Leu Ser Thr              20      #             25     #             30 Phe Asn Pro Cys Leu Ala Asn Met Glu Ala Ty#r Lys Lys Leu Glu Thr          35          #         40         #         45 Ile Phe Thr Lys Val Tyr Arg Leu Asp His Ph#e Leu Gly Leu Gly Asn      50              #     55             #     60 Trp Asp Met Asn Thr Asn Met Pro Pro Lys Gl#y Glu Glu Ser Arg Gly  65                  # 70                 # 75                  # 80 Glu Ala Met Ala Met Leu Ser Glu Leu Arg Ph#e Gly Phe Ile Thr Ala                  85  #                 90 #                 95 Pro Glu Val Lys Ser Leu Ile Glu Ser Ala Th#r Lys Gly Ser Glu Glu             100       #           105      #           110 Leu Asn Ala Val Gln Arg Ala Asn Leu Arg Gl#u Met Arg Arg Ala Trp         115           #       120          #       125 Lys Ser Ala Thr Ala Leu Pro Ala Glu Phe Va#l Gly Arg Lys Met Arg     130               #   135              #   140 Leu Thr Thr His Ala His Ser Val Trp Arg As#p Ser Arg Lys Ala Asn 145                 1 #50                 1#55                 1 #60 Asp Phe Ala Lys Phe Leu Pro Val Leu Arg As#p Leu Val Ala Leu Ala                 165   #               170  #               175 Arg Glu Glu Gly Ser Tyr Leu Ala Ala Gly Th#r Ser Leu Ser Pro Tyr             180       #           185      #           190 Glu Ala Leu Met Asn Glu Tyr Glu Pro Gly Il#e Thr Thr Gln Lys Leu         195           #       200          #       205 Asp Glu Val Tyr Ala Asn Val Lys Ser Trp Le#u Pro Gln Leu Leu Lys     210               #   215              #   220 Asp Ile Val Gln Lys Gln Ser Gly Glu Ser Va#l Ile Ala Phe Ser His 225                 2 #30                 2#35                 2 #40 Lys Phe Pro Gln Asp Lys Gln Glu Ala Leu Cy#s Lys Glu Phe Met Lys                 245   #               250  #               255 Ile Trp His Phe Asp Thr Asp Ala Gly Arg Le#u Asp Val Ser Pro His             260       #           265      #           270 Pro Phe Thr Gly Met Thr Lys Glu Asp Cys Ar#g Leu Thr Thr Asn Tyr         275           #       280          #       285 Ile Glu Asp Thr Phe Val Gln Ser Leu Tyr Gl#y Val Ile His Glu Ser     290               #   295              #   300 Gly His Gly Lys Tyr Glu Gln Asn Cys Gly Pr#o Arg Glu His Ile Thr 305                 3 #10                 3#15                 3 #20 Gln Pro Val Cys Asn Ala Arg Ser Leu Gly Le#u His Glu Ser Gln Ser                 325   #               330  #               335 Leu Phe Ala Glu Phe Gln Ile Gly His Ala Th#r Pro Phe Ile Asp Tyr             340       #           345      #           350 Leu Thr Thr Arg Leu Pro Glu Phe Phe Glu Al#a Gln Pro Ala Phe Ser         355           #       360          #       365 Gln Asp Asn Met Arg Lys Ser Leu Gln Gln Va#l Lys Pro Gly Tyr Ile     370               #   375              #   380 Arg Val Asp Ala Asp Glu Val Cys Tyr Pro Le#u His Val Ile Leu Arg 385                 3 #90                 3#95                 4 #00 Tyr Glu Ile Glu Arg Asp Leu Met Glu Gly Ly#s Met Glu Val Glu Asp                 405   #               410  #               415 Val Pro Arg Ala Trp Asn Ala Lys Met Gln Gl#u Tyr Leu Gly Leu Ser             420       #           425      #           430 Thr Glu Gly Arg Asp Asp Val Gly Cys Leu Gl#n Asp Val His Trp Ser         435           #       440          #       445 Met Gly Ala Leu Gly Tyr Phe Pro Thr Tyr Se#r Leu Gly Ala Met Tyr     450               #   455              #   460 Ala Ala Gln Ile Met Ala Ser Ile Arg Lys Gl#u Leu Gly Asp Asp Lys 465                 4 #70                 4#75                 4 #80 Val Asp Glu Cys Leu Arg Thr Gly Glu Leu Gl#y Pro Leu Leu Glu Lys                 485   #               490  #               495 Gln Gln Glu Lys Ile Trp Asp His Gly Cys Le#u Tyr Glu Thr Asp Asp             500       #           505      #           510 Leu Met Thr Arg Ala Thr Gly Glu Thr Leu As#n Pro Glu Tyr Leu Arg         515           #       520          #       525 Arg His Leu Glu Ala Arg Tyr Leu Asn Ala    530               #   535 <210> SEQ ID NO 119 <211> LENGTH: 193<212> TYPE: PRT <213> ORGANISM: Leishmania major and chagasi<400> SEQUENCE: 119 Met His His His His His His Asp Pro Pro Gl#y Cys Arg Asn Ser Ala                    #5                  #10                   #15 Arg Glu Leu Gln Gln Arg Leu Asp Thr Ala Th#r Gln Gln Arg Ala Glu              20      #             25     #             30 Leu Glu Ala Arg Val Ala Arg Leu Ala Ala As#p Arg Asp Glu Ala Arg          35          #         40         #         45 Gln Gln Leu Ala Ala Asn Ala Glu Glu Leu Gl#n Gln Arg Leu Asp Thr      50              #     55             #     60 Ala Thr Gln Gln Arg Ala Glu Leu Glu Ala Ar#g Val Ala Arg Leu Ala  65                  # 70                 # 75                  # 80 Ala Asp Gly Asp Glu Ala Arg Gln Gln Leu Al#a Ala Asn Ala Glu Glu                  85  #                 90 #                 95 Leu Gln Gln Arg Leu Asp Thr Ala Thr Gln Gl#n Arg Ala Glu Leu Glu             100       #           105      #           110 Ala Gln Val Ala Arg Leu Ala Ala Asn Ala Gl#u Glu Leu Gln Gln Arg         115           #       120          #       125 Leu Asp Thr Ala Thr Gln Gln Arg Ala Glu Le#u Glu Ala Arg Val Ala     130               #   135              #   140 Arg Leu Ala Ala Asp Arg Asp Glu Ala Arg Gl#n Gln Leu Ala Ala Asn 145                 1 #50                 1#55                 1 #60 Ala Glu Glu Leu Gln Gln Arg Leu Asp Thr Al#a Thr Gln Gln Arg Ala                 165   #               170  #               175 Glu Leu Glu Ala Gln Val Ala Arg Leu Ala Al#a Asn Ala Ala Arg Ala             180       #           185      #           190 Glu <210> SEQ ID NO 120 <211> LENGTH: 2310<212> TYPE: PRT <213> ORGANISM: Leishmania major and chagasi<400> SEQUENCE: 120 Met Ser Thr Pro Val Ser Gly Val Val Pro Gl#n Asp Arg Trp Gln Pro                    #5                  #10                   #15 Gln Gln Arg Val Lys Val Cys Gln Tyr Gln As#p Cys Gly Ala Pro Phe              20      #             25     #             30 Gly Phe Phe Ser Thr Lys Val Asn Cys His Ar#g Cys Gly Ile Val Leu          35          #         40         #         45 Cys Ser Lys Cys Ala Ala Thr Lys Thr Val Il#e Pro Arg Tyr Tyr Ser      50              #     55             #     60 Asn Glu Thr Val Pro Val Cys Gln Arg Cys Ty#r Gln Val Val Glu Arg  65                  # 70                 # 75                  # 80 Tyr Lys Glu Arg Gly Ser Val Thr Pro Gly Ty#r Val Val His Ser Thr                  85  #                 90 #                 95 Thr Ile Ser Ala Thr Pro Ala Arg Ser Ser Pr#o Val Pro Pro Leu His             100       #           105      #           110 Thr Thr Pro Ala Leu Arg Pro His Ala Pro Se#r Pro Gln Pro Ala Ser         115           #       120          #       125 Val Val Ser Thr Ala Thr Leu Val His Pro Va#l Glu Glu Asp Ala Val     130               #   135              #   140 Ser Thr Lys Pro Ser Val Ser Glu Ala Asp Le#u His Ala Leu Arg Ser 145                 1 #50                 1#55                 1 #60 Ile Ile Glu Thr Leu Gln Gln Ala Leu Asn As#p Glu Gln His Asn Ala                 165   #               170  #               175 Ala Leu Ala Ala Thr Ser Ala Ala Glu Gln Le#u Arg Thr Ala Lys Glu             180       #           185      #           190 Glu Asn Thr Ala Leu Lys Ser Thr Ala His Le#u Leu Gln Gln Arg Leu         195           #       200          #       205 Asp Thr Ala Thr Gln Gln Arg Ala Glu Leu Gl#u Ala Gln Val Ala Arg     210               #   215              #   220 Leu Ala Ala Asp Arg Asp Glu Ala Arg Gln Gl#n Leu Ala Ala Asn Ala 225                 2 #30                 2#35                 2 #40 Glu Glu Leu Gln Gln Arg Leu Asp Thr Ala Th#r Gln Gln Arg Ala Glu                 245   #               250  #               255 Leu Glu Ala Arg Val Ala Arg Leu Ala Ala As#p Arg Asp Glu Ala Arg             260       #           265      #           270 Gln Gln Leu Ala Ala Asn Ala Glu Glu Leu Gl#n Gln Arg Leu Asp Thr         275           #       280          #       285 Ala Thr Gln Gln Arg Ala Glu Leu Glu Ala Gl#n Val Ala Arg Leu Ala     290               #   295              #   300 Ala Asn Ala Glu Glu Leu Gln Gln Arg Leu As#p Thr Ala Thr Gln Gln 305                 3 #10                 3#15                 3 #20 Arg Ala Glu Leu Glu Ala Gln Leu Ala Arg Le#u Ala Ala Asp Arg Asp                 325   #               330  #               335 Glu Ala Arg Gln Gln Leu Ala Ala Asn Ala Gl#u Glu Leu Gln Gln Arg             340       #           345      #           350 Leu Asp Thr Ala Thr Gln Gln Arg Ala Glu Le#u Glu Ala Gln Val Ala         355           #       360          #       365 Arg Leu Ala Ala Asn Ala Glu Glu Leu Gln Gl#n Arg Leu Asp Thr Ala     370               #   375              #   380 Thr Gln Gln Arg Ala Glu Leu Glu Ala Arg Va#l Ala Arg Leu Ala Ala 385                 3 #90                 3#95                 4 #00 Asp Arg Asp Glu Ala Arg Gln Gln Leu Ala Al#a Asn Ala Glu Glu Leu                 405   #               410  #               415 Gln Gln Arg Leu Asp Thr Ala Thr Gln Gln Ar#g Ala Glu Leu Glu Ala             420       #           425      #           430 Gln Val Ala Arg Leu Ala Ala Asn Arg Asp Gl#u Ala Arg Gln Gln Leu         435           #       440          #       445 Ala Ala Asn Ala Glu Glu Leu Gln Gln Arg Le#u Asp Thr Ala Thr Gln     450               #   455              #   460 Gln Arg Ala Glu Leu Glu Ala Gln Val Ala Ar#g Leu Ala Ala Asp Arg 465                 4 #70                 4#75                 4 #80 Asp Glu Ala Arg Gln Gln Leu Ala Ala Asn Al#a Glu Glu Leu Gln Gln                 485   #               490  #               495 Arg Leu Asp Thr Ala Thr Gln Gln Arg Ala Gl#u Leu Glu Ala Arg Val             500       #           505      #           510 Ala Arg Leu Ala Ala Asp Arg Asp Glu Ala Ar#g Gln Gln Leu Ala Ala         515           #       520          #       525 Asn Ala Glu Glu Leu Gln Gln Arg Leu Asp Th#r Ala Thr Gln Gln Arg     530               #   535              #   540 Ala Glu Leu Glu Ala Arg Val Ala Arg Leu Al#a Ala Asn Ala Glu Glu 545                 5 #50                 5#55                 5 #60 Leu Gln Gln Arg Leu Asp Thr Ala Thr Gln Gl#n Arg Ala Glu Leu Glu                 565   #               570  #               575 Ala Gln Val Ala Arg Leu Ala Ala Asn Ala Gl#u Glu Leu Gln Gln Arg             580       #           585      #           590 Leu Asp Thr Ala Thr Gln Gln Arg Ala Glu Le#u Glu Ala Arg Val Ala         595           #       600          #       605 Arg Leu Ala Ala Asp Arg Asp Glu Ala Arg Gl#n Gln Leu Ala Ala Asn     610               #   615              #   620 Ala Glu Glu Leu Gln Gln Arg Leu Asp Thr Al#a Thr Gln Gln Arg Ala 625                 6 #30                 6#35                 6 #40 Glu Leu Glu Ala Gln Leu Ala Arg Leu Ala Al#a Asp Gly Asp Glu Ala                 645   #               650  #               655 Arg Gln Gln Leu Ala Ala Asn Ala Glu Glu Le#u Gln Gln Arg Leu Asp             660       #           665      #           670 Thr Ala Thr Gln Gln Arg Ala Glu Leu Glu Al#a Arg Val Ala Arg Leu         675           #       680          #       685 Ala Ala Asp Arg Asp Glu Ala Arg Gln Gln Le#u Ala Ala Asn Ala Glu     690               #   695              #   700 Glu Leu Gln Gln Arg Leu Asp Thr Ala Thr Gl#n Gln Arg Ala Glu Leu 705                 7 #10                 7#15                 7 #20 Glu Ala Gln Leu Ala Arg Leu Ala Ala Asp Ar#g Asp Glu Ala Arg Gln                 725   #               730  #               735 Gln Leu Ala Ala Asn Ala Glu Glu Leu Gln Gl#n Arg Leu Asp Thr Ala             740       #           745      #           750 Thr Gln Gln Arg Ala Glu Leu Glu Ala Arg Va#l Ala Arg Leu Ala Ala         755           #       760          #       765 Asp Gly Asp Glu Ala Arg Gln Gln Leu Ala Al#a Asn Ala Glu Glu Leu     770               #   775              #   780 Gln Gln Arg Leu Asp Thr Ala Thr Gln Gln Ar#g Ala Glu Leu Glu Ala 785                 7 #90                 7#95                 8 #00 Arg Val Ala Arg Leu Ala Ala Asp Arg Asp Gl#u Ala Arg Gln Gln Leu                 805   #               810  #               815 Ala Ala Asn Ala Glu Glu Leu Gln Gln Arg Le#u Asp Thr Ala Thr Gln             820       #           825      #           830 Gln Arg Ala Glu Leu Glu Ala Gln Leu Ala Ar#g Leu Ala Ala Asp Gly         835           #       840          #       845 Asp Glu Ala Arg Gln Gln Leu Ala Ala Asn Al#a Glu Glu Leu Gln Gln     850               #   855              #   860 Arg Leu Asp Thr Ala Thr Gln Gln Arg Ala Gl#u Leu Glu Ala Arg Val 865                 8 #70                 8#75                 8 #80 Ala Arg Leu Ala Ala Asp Arg Asp Glu Ala Ar#g Gln Gln Leu Ala Ala                 885   #               890  #               895 Asn Ala Glu Glu Leu Gln Gln Arg Leu Asp Th#r Ala Thr Gln Gln Arg             900       #           905      #           910 Ala Glu Leu Glu Ala Gln Leu Ala Arg Leu Al#a Ala Asp Arg Asp Glu         915           #       920          #       925 Ala Arg Gln Gln Leu Ala Ala Asn Ala Glu Gl#u Leu Gln Gln Arg Leu     930               #   935              #   940 Asp Thr Ala Thr Gln Gln Arg Ala Glu Leu Gl#u Ala Gln Val Ala Arg 945                 9 #50                 9#55                 9 #60 Leu Ala Ala Asp Gly Asp Glu Ala Arg Gln Gl#n Leu Ala Ala Asn Ala                 965   #               970  #               975 Glu Glu Leu Gln Gln Arg Leu Asp Thr Ala Th#r Gln Gln Arg Ala Glu             980       #           985      #           990 Leu Glu Ala Gln Leu Ala Arg Leu Ala Ala As#p Arg Asp Glu Ala Arg         995           #       1000          #      1005 Gln Gln Leu Ala Ala Asn Ala Glu Glu Leu Gl#n Gln Arg Leu Asp Thr     1010              #   1015              #  1020 Ala Thr Gln Gln Arg Ala Glu Leu Glu Ala Gl#n Val Ala Arg Leu Ala 1025                1030 #                1035 #               1040 Ala Asn Ala Glu Glu Leu Gln Gln Arg Leu As#p Thr Ala Thr Gln Gln                 1045  #               1050  #              1055 Arg Ala Glu Leu Glu Ala Arg Val Ala Arg Le#u Ala Ala Asp Arg Asp             1060      #           1065      #          1070 Glu Ala Arg Gln Gln Leu Ala Ala Asn Ala Gl#u Glu Leu Gln Gln Arg         1075          #       1080          #      1085 Leu Asp Thr Ala Thr Gln Gln Arg Ala Glu Le#u Glu Ala Arg Val Ala     1090              #   1095              #  1100 Arg Leu Ala Ala Asn Ala Glu Glu Leu Gln Gl#n Arg Leu Asp Thr Ala 1105                1110 #                1115 #               1120 Thr Gln Gln Arg Ala Glu Leu Glu Ala Gln Va#l Ala Arg Leu Ala Ala                 1125  #               1130  #              1135 Asn Ala Glu Glu Leu Gln Gln Arg Leu Asp Th#r Ala Thr Gln Gln Arg             1140      #           1145      #          1150 Ala Glu Leu Glu Ala Arg Val Ala Arg Leu Al#a Ala Asp Arg Asp Glu         1155          #       1160          #      1165 Ala Arg Gln Gln Leu Ala Ala Asn Ala Glu Gl#u Leu Gln Gln Arg Leu     1170              #   1175              #  1180 Asp Thr Ala Thr Gln Gln Arg Ala Glu Leu Gl#u Ala Gln Val Ala Arg 1185                1190 #                1195 #               1200 Leu Ala Ala Asn Ala Glu Glu Leu Gln Gln Ar#g Leu Asp Thr Ala Thr                 1205  #               1210  #              1215 Gln Gln Arg Ala Glu Leu Glu Ala Gln Leu Al#a Arg Leu Ala Ala Asp             1220      #           1225      #          1230 Arg Asp Glu Ala Arg Gln Gln Leu Ala Ala As#n Ala Glu Glu Leu Gln         1235          #       1240          #      1245 Gln Arg Leu Asp Thr Ala Thr Gln Gln Arg Al#a Glu Leu Glu Ala Gln     1250              #   1255              #  1260 Val Ala Arg Leu Ala Ala Asn Ala Glu Glu Le#u Gln Gln Arg Leu Asp 1265                1270 #                1275 #               1280 Thr Ala Thr Gln Gln Arg Ala Glu Leu Glu Al#a Arg Val Ala Arg Leu                 1285  #               1290  #              1295 Ala Ala Asp Arg Asp Glu Ala Arg Gln Gln Le#u Ala Ala Asn Ala Glu             1300      #           1305      #          1310 Glu Leu Gln Gln Arg Leu Asp Thr Ala Thr Gl#n Gln Arg Ala Glu Leu         1315          #       1320          #      1325 Glu Ala Arg Val Ala Arg Leu Ala Ala Asp Gl#y Asp Glu Ala Arg Gln     1330              #   1335              #  1340 Gln Leu Ala Ala Asn Ala Glu Glu Leu Gln Gl#n Arg Leu Asp Thr Ala 1345                1350 #                1355 #               1360 Thr Gln Gln Arg Ala Glu Leu Glu Ala Gln Va#l Ala Arg Leu Ala Ala                 1365  #               1370  #              1375 Asn Ala Glu Glu Leu Gln Gln Arg Leu Asp Th#r Ala Thr Gln Gln Arg             1380      #           1385      #          1390 Ala Glu Leu Glu Ala Arg Val Ala Arg Leu Al#a Ala Asp Arg Asp Glu         1395          #       1400          #      1405 Ala Arg Gln Gln Leu Ala Ala Asn Ala Glu Gl#u Leu Gln Gln Arg Leu     1410              #   1415              #  1420 Asp Thr Ala Thr Gln Gln Arg Ala Glu Leu Gl#u Ala Gln Val Ala Arg 1425                1430 #                1435 #               1440 Leu Ala Ala Asp Arg Asp Glu Ala Arg Gln Gl#n Leu Ala Ala Asn Ala                 1445  #               1450  #              1455 Glu Glu Leu Gln Gln Arg Leu Asp Thr Ala Th#r Gln Gln Arg Ala Glu             1460      #           1465      #          1470 Leu Glu Ala Arg Val Ala Arg Leu Ala Ala As#p Gly Asp Glu Ala Arg         1475          #       1480          #      1485 Gln Gln Leu Ala Ala Asn Ala Glu Glu Leu Gl#n Gln Arg Leu Asp Thr     1490              #   1495              #  1500 Ala Thr Gln Gln Arg Ala Glu Leu Glu Ala Gl#n Leu Ala Arg Leu Ala 1505                1510 #                1515 #               1520 Ala Asp Arg Asp Glu Ala Arg Gln Gln Leu Al#a Ala Asn Ala Glu Glu                 1525  #               1530  #              1535 Leu Gln Gln Arg Leu Asp Thr Ala Thr Gln Gl#n Arg Ala Glu Leu Glu             1540      #           1545      #          1550 Ala Arg Val Ala Arg Leu Ala Ala Asp Gly As#p Glu Ala Arg Gln Gln         1555          #       1560          #      1565 Leu Ala Ala Asn Ala Glu Glu Leu Gln Gln Ar#g Leu Asp Thr Ala Thr     1570              #   1575              #  1580 Gln Gln Arg Ala Glu Leu Glu Ala Arg Val Al#a Arg Leu Ala Ala Asp 1585                1590 #                1595 #               1600 Arg Asp Glu Ala Arg Gln Gln Leu Ala Ala As#n Ala Glu Glu Leu Gln                 1605  #               1610  #              1615 Gln Arg Leu Asp Thr Ala Thr Gln Gln Arg Al#a Glu Leu Glu Ala Gln             1620      #           1625      #          1630 Leu Ala Arg Leu Ala Ala Asp Arg Asp Glu Al#a Arg Gln Gln Leu Ala         1635          #       1640          #      1645 Ala Asn Ala Glu Glu Leu Gln Gln Arg Leu As#p Thr Ala Thr Gln Gln     1650              #   1655              #  1660 Arg Ala Glu Leu Glu Ala Gln Leu Ala Arg Le#u Ala Ala Asp Gly Asp 1665                1670 #                1675 #               1680 Glu Ala Arg Gln Gln Leu Ala Ala Asn Ala Gl#u Glu Leu Gln Gln Arg                 1685  #               1690  #              1695 Leu Asp Thr Ala Thr Gln Gln Arg Ala Glu Le#u Glu Val Glu Met Ala             1700      #           1705      #          1710 Val Leu Leu Arg Glu Arg Glu Glu Ala Arg Gl#y Glu Thr Ala Val Ala         1715          #       1720          #      1725 Gly Glu Gln Val Gln Leu Tyr Arg Glu Thr Va#l Glu Glu Glu Glu Cys     1730              #   1735              #  1740 Leu Lys Glu Glu Arg Trp Cys Leu Glu Ser Ar#g Val Ala Gln Leu Arg 1745                1750 #                1755 #               1760 Glu Ala Ser Ala Ala Ala Lys Gln Gln Arg Gl#n Glu Val Ala Ala Lys                 1765  #               1770  #              1775 Ala Asn Glu Val Gln Glu Arg Leu Asp Ser Me#t Ala Arg Arg Cys Ile             1780      #           1785      #          1790 Ala His Glu Gly Asp Ala Pro Gln Arg Ala As#p Gly Arg Asp Asp Ala         1795          #       1800          #      1805 Leu Arg Gln Leu Ala Asn Leu Arg Glu Glu Va#l Lys Leu Ser Glu Lys     1810              #   1815              #  1820 Gln Lys Ala Met Glu Arg Val Ile Pro Gly Va#l Arg Glu Arg Gln Met 1825                1830 #                1835 #               1840 Arg Leu Glu Ala Ala Glu Glu Gln Arg Ala As#p Leu Glu Ala Arg Leu                 1845  #               1850  #              1855 Val Asp Glu Ala Gly Asp Leu Arg Ser Arg Pr#o Ala Ala Ser Thr Asn             1860      #           1865      #          1870 Glu Val Asn Leu Tyr Arg Asp Leu Ala Leu Gl#n Glu His Glu Ala Ala         1875          #       1880          #      1885 Gln Asn Arg Cys Thr Thr Leu Glu Ala Gln Va#l Ala Ser Leu Thr Ser     1890              #   1895              #  1900 Asp Arg Asp Asn Gly Arg Gln Gln Glu Ser Al#a Asp Leu Ser Glu Ala 1905                1910 #                1915 #               1920 Gln Arg His Leu Asp Asn Val Gln Glu Arg As#p Met Ala His His Arg                 1925  #               1930  #              1935 Cys Ala Ala Leu Glu Glu Gln Asn Ala Ala Me#t Ala Ser Glu Leu Gln             1940      #           1945      #          1950 Ala Val Lys Ala Lys Leu Arg Gln Ala Ser Va#l Lys Ala Ser Ser Leu         1955          #       1960          #      1965 Met Thr Arg Leu Ser Ala Ser Ser Ser Gly Al#a Gly Gly Val Ser Ala     1970              #   1975              #  1980 Arg Val Arg Val Gly Gly Ser Ser Ala Val Pr#o Gln Ala Ala Pro His 1985                1990 #                1995 #               2000 Arg Asp Ala Glu Leu Ile Ala Glu Val Gly Gl#u Arg Leu Arg Glu Arg                 2005  #               2010  #              2015 Gly Glu Ala Met Arg Leu Leu Ala Glu Gly Va#l Glu Leu Arg Glu Arg             2020      #           2025      #          2030 Ala Arg Pro Leu Glu Arg Val Leu Ala Glu Ly#s Leu Ile Gly Asp Arg         2035          #       2040          #      2045 Arg Thr Ser Asp Ala Glu Glu Val Ala Thr Gl#u Pro Thr Gln Val Arg     2050              #   2055              #  2060 Arg Asn Ala Ala His Ser Arg His Leu Asp Se#r Arg Glu Ala Gln Leu 2065                2070 #                2075 #               2080 Asp Glu Arg Ala Ala Arg Leu Arg Glu Lys Gl#u Gln Gln Leu Leu Arg                 2085  #               2090  #              2095 Val Ala Arg Glu Leu Gln Thr Lys Ser Arg Al#a Leu Gln Val Leu Tyr             2100      #           2105      #          2110 Ala Arg Ala Leu Asn Arg Pro Gln Val Thr Se#r Leu Leu Leu Thr Ala         2115          #       2120          #      2125 Asp Gly Asp Asp Thr Ser Tyr Pro Asp Thr Pr#o Gln Gln Gln Gln Gln     2130              #   2135              #  2140 Gly Thr Arg Thr Pro Leu Arg Glu Pro Val Ty#r Ser Leu Asp Ser Glu 2145                2150 #                2155 #               2160 Val Ala His Tyr Gly Arg Thr Ala Gly Ala Al#a Val Ser Ser Gly Leu                 2165  #               2170  #              2175 Ala Ser Pro Leu Pro Arg Glu Pro Pro Arg Al#a Arg Met Val His Arg             2180      #           2185      #          2190 Ala Val Glu Ala Thr Gly Thr Glu Glu Asp Th#r Gln Val Arg Leu Thr         2195          #       2200          #      2205 Ala Ala Thr Glu Ala Tyr Arg Asp Val Leu Ty#r Glu His Ile Leu Glu     2210              #   2215              #  2220 Ser Asn Gly Leu Gln Gly Val Asp Val Leu Al#a Gln Tyr Leu Pro His 2225                2230 #                2235 #               2240 His Thr Ser Gly Gly Gly Leu Lys Thr Pro Ar#g Leu Pro Gly Ser Gly                 2245  #               2250  #              2255 Ile Ile Ser Lys Thr Arg Ala Met Leu Arg Al#a Leu Glu Glu Arg Leu             2260      #           2265      #          2270 Gly Ala Ser Arg Gly Val Gly Arg Gly Val As#p Pro Ala Val Gln Glu         2275          #       2280          #      2285 Arg Ser Leu Glu Ala Phe Arg Arg Leu Glu Al#a Ala Leu Ser Ala Leu     2290              #   2295              #  2300 Cys Gly Gly Ser His Ala 2305                2310<210> SEQ ID NO 121 <211> LENGTH: 709 <212> TYPE: PRT<213> ORGANISM: Leishmania major and chagasi <400> SEQUENCE: 121Met Arg Ser Ser Arg Gly Gly Ser Leu Ala Va #l Val Ala Leu Ala Val                   #5                   #10                   #15Cys Leu Ala Val Leu Ala Ala Ile Gly Thr Cy #s Val Phe Asp Ser Gln             20      #             25      #             30Glu Ile Gly Gly Ser Ser Phe Thr Phe Val Gl #y Trp Ser Ser Ala Ser         35          #         40          #         45Lys Glu Glu Ser Tyr Gln Gly Cys Thr Leu Th #r Gly Lys Ala Phe Arg     50              #     55              #     60Ile Gln Gly Ala Ala Ser Ser Leu Ser Asp As #p Ala Thr Leu Pro Gly 65                  # 70                  # 75                  # 80Gly Ile Leu Arg Phe Ser Ser Leu Leu Val Se #r Asn Gly Tyr Ile Val                 85  #                 90  #                 95Val Asp Lys Tyr Phe Pro Arg Asn Thr Asn Il #e Thr Ile Lys Asp Ala            100       #           105       #           110Ser Gly Thr Val Ala Ala Gly Met Pro Phe Il #e Asp Ala Asn Thr Ala        115           #       120           #       125Ile Tyr Ser Asp Gln Leu Ser Ile Val Val Th #r Asp Ser Thr Leu Ser    130               #   135               #   140Trp Ala Ala Ala Arg Ser Gly Gln Ser Met Va #l Arg Ala Pro Phe Thr145                 1 #50                 1 #55                 1 #60Ile Gln Leu Ser Ser Ser Leu Phe Val Leu Gl #y Ser Thr Val Thr Gln                165   #               170   #               175Ala Ser Ser Val Val Glu Val Val Gly Pro Se #r Ser Ile Ser Gln Lys            180       #           185       #           190Ser Ala Leu Ala Val Asp Tyr Ala Lys Cys Th #r Gly Cys Ala Gln Gly        195           #       200           #       205Leu Val His Phe Thr Asp Phe Val Arg Val Tr #p Asp Arg Ser Leu Leu    210               #   215               #   220Arg Val Ser His Ser Ser Val Lys Gly Ala Th #r Gly Lys Pro Leu Ile225                 2 #30                 2 #35                 2 #40Gly Ile Ala Gln Ser Ala Gly Ala Ser Leu Al #a Val Glu Asn Ser Leu                245   #               250   #               255Phe Val Val Glu Asn Val Ser Ser Pro Thr Se #r Asn Leu Ile Asp Ala            260       #           265       #           270Ala Val Arg Met Gly Thr Asp Ala Gln Ile Th #r Leu Arg Ala Val Thr        275           #       280           #       285Val Lys Ser Ile Gly Ala Thr Met Ala Gly Se #r Val Thr Ala Gln Leu    290               #   295               #   300Leu Thr Ala Asp Asp Ile Ala Gln Gln Ile Pr #o Ser Ile Ser Val Val305                 3 #10                 3 #15                 3 #20Pro Asp Thr Arg Cys Ala Ala Ala Cys Val Pr #o Thr Ala Thr Val Asp                325   #               330   #               335Ser Ser Cys Lys Cys Ala Cys Asn Ala Asp Me #t Pro Asn Met Asn Phe            340       #           345       #           350Cys Thr Ala Met Lys Asp Pro Tyr Thr Asn Ty #r Ala Tyr Leu Gly Cys        355           #       360           #       365Ser Ala Gly Cys Thr Thr Cys Phe Asn Glu Th #r Ala Cys Leu Glu Cys    370               #   375               #   380Arg Pro Ser Tyr Glu Met Leu Pro Asp Met Th #r Cys Ser Leu Thr Gly385                 3 #90                 3 #95                 4 #00Leu Gln Cys Thr Asp Pro Asn Cys Lys Thr Cy #s Thr Thr Tyr Gly Gln                405   #               410   #               415Cys Thr Asp Cys Asn Asp Gly Tyr Gly Leu Th #r Ser Ser Ser Val Cys            420       #           425       #           430Val Arg Cys Ser Val Ala Gly Cys Lys Ser Cy #s Pro Val Asp Ala Asn        435           #       440           #       445Val Cys Lys Val Cys Leu Gly Gly Ser Glu Pr #o Ile Asn Asn Met Cys    450               #   455               #   460Pro Cys Thr Asp Pro Asn Cys Ala Ser Cys Pr #o Ser Asp Ala Gly Thr465                 4 #70                 4 #75                 4 #80Cys Thr Gln Cys Ala Asn Gly Tyr Gly Leu Va #l Asp Gly Ala Cys Val                485   #               490   #               495Arg Cys Gln Glu Pro Asn Cys Phe Ser Cys As #p Ser Asp Ala Asn Lys            500       #           505       #           510Cys Thr Gln Cys Ala Pro Asn Tyr Tyr Leu Th #r Pro Leu Leu Thr Cys        515           #       520           #       525Ser Pro Val Ala Cys Asn Ile Glu His Cys Me #t Gln Cys Asp Pro Gln    530               #   535               #   540Thr Pro Ser Arg Cys Gln Glu Cys Val Ser Pr #o Tyr Val Val Asp Ser545                 5 #50                 5 #55                 5 #60Tyr Asp Gly Leu Cys Arg Leu Ser Asp Ala Cy #s Ser Val Pro Asn Cys                565   #               570   #               575Lys Lys Cys Glu Thr Gly Thr Ser Arg Leu Cy #s Ala Glu Cys Asp Thr            580       #           585       #           590Gly Tyr Ser Leu Ser Ala Asp Ala Thr Ser Cy #s Ser Ser Pro Thr Thr        595           #       600           #       605Gln Pro Cys Glu Val Glu His Cys Asn Thr Cy #s Val Asn Gly Asp Ser    610               #   615               #   620Thr Arg Cys Ala Tyr Cys Asn Thr Gly Tyr Ty #r Val Ser Asp Gly Lys625                 6 #30                 6 #35                 6 #40Cys Lys Ala Met Gln Gly Cys Tyr Val Ser As #n Cys Ala Gln Cys Met                645   #               650   #               655Leu Leu Asp Ser Thr Lys Cys Ser Thr Cys Va #l Lys Gly Tyr Leu Leu            660       #           665       #           670Thr Ser Ser Tyr Ser Cys Val Ser Gln Lys Va #l Ile Asn Ser Ala Ala        675           #       680           #       685Ala Pro Tyr Ser Leu Trp Val Ala Ala Ala Va #l Leu Leu Thr Ser Phe    690               #   695               #   700 Ala Met His Leu Ala705 <210> SEQ ID NO 122 <211> LENGTH: 302 <212> TYPE: PRT<213> ORGANISM: Leishmania major and chagasi <400> SEQUENCE: 122Met His His His His His His Asp Ser Gln Gl #u Ile Gly Gly Ser Ser                   #5                   #10                   #15Phe Thr Phe Val Gly Trp Ser Ser Ala Ser Ly #s Glu Glu Ser Tyr Gln             20      #             25      #             30Gly Cys Thr Leu Thr Gly Lys Ala Phe Arg Il #e Gln Gly Ala Ala Ser         35          #         40          #         45Ser Leu Ser Asp Asp Ala Thr Leu Pro Gly Gl #y Ile Leu Arg Phe Ser     50              #     55              #     60Ser Leu Leu Val Ser Asn Gly Tyr Ile Val Va #l Asp Lys Tyr Phe Pro 65                  # 70                  # 75                  # 80Arg Asn Thr Asn Ile Thr Ile Lys Asp Ala Se #r Gly Thr Val Ala Ala                 85  #                 90  #                 95Gly Met Pro Phe Ile Asp Ala Asn Thr Ala Il #e Tyr Ser Asp Gln Leu            100       #           105       #           110Ser Ile Val Val Thr Asp Ser Thr Leu Ser Tr #p Ala Ala Ala Arg Ser        115           #       120           #       125Gly Gln Ser Met Val Arg Ala Pro Phe Thr Il #e Gln Leu Ser Ser Ser    130               #   135               #   140Leu Phe Val Leu Gly Ser Thr Val Thr Gln Al #a Ser Ser Val Val Glu145                 1 #50                 1 #55                 1 #60Val Val Gly Pro Ser Ser Ile Ser Gln Lys Se #r Ala Leu Ala Val Asp                165   #               170   #               175Tyr Ala Lys Cys Thr Gly Cys Ala Gln Gly Le #u Val His Phe Thr Asp            180       #           185       #           190Phe Val Arg Val Trp Asp Arg Ser Leu Leu Ar #g Val Ser His Ser Ser        195           #       200           #       205Val Lys Gly Ala Thr Gly Lys Pro Leu Ile Gl #y Ile Ala Gln Ser Ala    210               #   215               #   220Gly Ala Ser Leu Ala Val Glu Asn Ser Leu Ph #e Val Val Glu Asn Val225                 2 #30                 2 #35                 2 #40Ser Ser Pro Thr Ser Asn Leu Ile Asp Ala Al #a Val Arg Met Gly Thr                245   #               250   #               255Asp Ala Gln Ile Thr Leu Arg Ala Val Thr Va #l Lys Ser Ile Gly Ala            260       #           265       #           270Thr Met Ala Gly Ser Val Thr Ala Gln Leu Le #u Thr Ala Asp Asp Ile        275           #       280           #       285Ala Gln Gln Ile Pro Ser Ile Ser Val Val Pr #o Asp Thr Arg    290               #   295               #   300

What is claimed is:
 1. A method for inducing protective immunity againstleishmaniasis in a patient comprising administering an immunogeniccomposition comprising a fusion protein and an immunostimulant, whereinthe fusion protein comprises the amino acid sequence of SEQ ID NO:24. 2.The method of claim 1, wherein the fusion protein comprises the aminoacid sequence of SEQ ID NO:95.
 3. The method of any one of claims 1 and2, wherein the immunostimulant is selected from the group consisting of:aminoalkyl glucosaminide 4-phosphates; monophosphoryl lipid A; and3-de-O-acylated monophosphoryl lipid A.